Specific binding proteins and uses thereof

ABSTRACT

The invention relates to specific binding members, particularly antibodies and active fragments thereof, which recognize an aberrant post-translationally modified, particularly an aberrant glycosylated form of the EGFR. The binding members, particularly antibodies and fragments thereof, of the invention do not bind to EGFR on normal cells in the absence of amplification of the wild-type gene and are capable of binding the de2-7 EGFR at an epitope which is distinct from the junctional peptide. Antibodies of this type are exemplified by the novel antibody 806 whose VH and VL sequences are illustrated as SEQ ID NOs: 2 and 4 and chimeric antibodies thereof as exemplified by ch806.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/317,683, filed Dec. 23, 2008, now abandoned; which is a continuationof U.S. application Ser. No. 10/145,598, filed May 13, 2002, now U.S.Pat. No. 7,589,180, issued Sep. 15, 2009; which claims priority to U.S.Provisional Application No. 60/290,410, filed May 11, 2001, U.S.Provisional Application No. 60/326,019, filed Sep. 28, 2001, and U.S.Provisional Application No. 60/342,258, filed Dec. 21, 2001.

FIELD OF THE INVENTION

The present invention relates to specific binding members, particularlyantibodies and fragments thereof, which bind to amplified epidermalgrowth factor receptor (EGFR) and to the de2-7 EGFR truncation of theEGFR. In particular, the epitope recognized by the specific bindingmembers, particularly antibodies and fragments thereof, is enhanced orevident upon aberrant post-translational modification. These specificbinding members are useful in the diagnosis and treatment of cancer. Thebinding members of the present invention may also be used in therapy incombination with chemotherapeutics or anti-cancer agents and/or withother antibodies or fragments thereof.

BACKGROUND OF THE INVENTION

The treatment of proliferative disease, particularly cancer, bychemotherapeutic means often relies upon exploiting differences intarget proliferating cells and other normal cells in the human or animalbody. For example, many chemical agents are designed to be taken up byrapidly replicating DNA so that the process of DNA replication and celldivision is disrupted. Another approach is to identify antigens on thesurface of tumor cells or other abnormal cells which are not normallyexpressed in developed human tissue, such as tumor antigens or embryonicantigens. Such antigens can be targeted with binding proteins such asantibodies which can block or neutralize the antigen. In addition, thebinding proteins, including antibodies and fragments thereof, maydeliver a toxic agent or other substance which is capable of directly orindirectly activating a toxic agent at the site of a tumor.

The EGFR is an attractive target for tumor-targeted antibody therapybecause it is over-expressed in many types of epithelial tumors (27,28). Moreover, expression of the EGFR is associated with poor prognosisin a number of tumor types including stomach, colon, urinary bladder,breast, prostate, endometrium, kidney and brain (e.g., glioma).Consequently, a number of EGFR antibodies have been reported in theliterature with several undergoing clinical evaluation (18, 19, 29).Results from studies using EGFR mAbs in patients with head and neckcancer, squamous cell lung cancer, brain gliomas and malignantastrocytomas have been encouraging. The anti-tumor activity of most EGFRantibodies is enhanced by their ability to block ligand binding (30,31). Such antibodies may mediate their efficacy through both modulationof cellular proliferation and antibody dependent immune functions (e.g.complement activation). The use of these antibodies, however, may belimited by uptake in organs that have high endogenous levels of EGFRsuch as the liver and skin (18, 19).

A significant proportion of tumors containing amplifications of the EGFRgene (i.e., multiple copies of the EGFR gene) also co-express atruncated version of the receptor (13) known as de2-7 EGFR, ΔEGFR, orΔ2-7 (terms used interchangeably herein) (2). The rearrangement seen inthe de2-7 EGFR results in an in-frame mature mRNA lacking 801nucleotides spanning exons 2-7 (6-9). The corresponding EGFR protein hasa 267 amino acid deletion comprising residues 6-273 of the extracellulardomain and a novel glycine residue at the fusion junction (9). Thisdeletion, together with the insertion of a glycine residue, produces aunique junctional peptide at the deletion interface (9). The de2-7 EGFR(2) has been reported in a number of tumor types including glioma,breast, lung, ovarian and prostate (1-4). While this truncated receptordoes not bind ligand, it possesses low constitutive activity and impartsa significant growth advantage to glioma cells grown as tumor xenograftsin nude mice (10) and is able to transform NIH3T3 cells (11) and MCF-7cells. The cellular mechanisms utilized by the de2-7 EGFR in gliomacells are not fully defined but are reported to include a decrease inapoptosis (12) and a small enhancement of proliferation (12).

As expression of this truncated receptor is restricted to tumor cells itrepresents a highly specific target for antibody therapy. Accordingly, anumber of laboratories have reported the generation of both polyclonal(14) and monoclonal (3, 15, 16) antibodies specific to the uniquepeptide of de2-7 EGFR. A series of mouse mAbs, isolated followingimmunization with the unique de2-7 peptide, all showed selectivity andspecificity for the truncated receptor and targeted de2-7 EGFR positivexenografts grown in nude mice (3, 25, 32).

However, one potential shortcoming of de2-7 EGFR antibodies is that onlya proportion of tumors exhibiting amplification of the EGFR gene alsoexpress the de 2-7 EGFR (5). The exact percentage of tumors containingthe de2-7 EGFR is not completely established, because the use ofdifferent techniques (i.e. PCR versus immunohistochemistry) and variousantibodies, has produced a wide range of reported values for thefrequency of its presence. Published data indicates that approximately25-30% of gliomas express de2-7 EGFR with expression being lowest inanaplastic astrocytomas and highest in glioblastoma multiforme(6,13,17). The proportion of positive cells within de2-7 EGFR expressinggliomas has been reported to range from 37-86% (1). 27% of breastcarcinomas and 17% of lung cancers were found to be positive for thede2-7 EGFR (1, 3, 13, 16). Thus, de2-7 EGFR specific antibodies would beexpected to be useful in only a percentage of EGFR positive tumors.

Thus, while the extant evidence of activity of EGFR antibodies isencouraging, the observed limitations on range of applicability andefficacy reflected above remain.

Accordingly, it would be desirable to develop antibodies and like agentsthat demonstrate efficacy with a broad range of tumors, and it is towardthe achievement of that objective that the present invention isdirected.

The citation of references herein shall not be construed as an admissionthat such is prior art to the present invention.

SUMMARY OF THE INVENTION

In a broad aspect, the present invention provides an isolated specificbinding member, particularly an antibody or fragment thereof, whichrecognizes an EGFR epitope which does not demonstrate any amino acidsequence alterations or substitutions from wild type EGFR and which isfound in tumorigenic, hyperproliferative or abnormal cells and is notdetectable in normal or wild type cells (the term “wild type cell” asused herein contemplates a cell that expresses endogenous EGFR but notthe de 2-7 EGFR and the term specifically excludes a cell thatoverexpresses the EGFR gene; the term “wild type” refers to a genotypeor phenotype or other characteristic present in a normal cell ratherthan in an abnormal or tumorigenic cell). In a further aspect, thepresent invention provides a specific binding member, particularly anantibody or fragment thereof, which recognizes an EGFR epitope which isfound in tumorigenic, hyperproliferative or abnormal cells and is notdetectable in normal or wild type cells, wherein the epitope is enhancedor evident upon aberrant post translational modification or aberrantexpression. In a particular nonlimiting exemplification provided herein,the EGFR epitope is enhanced or evident wherein post-translationalmodification is not complete or full to the extent seen with normalexpression of EGFR in wild type cells. In one aspect, the EGFR epitopeis enhanced or evident upon initial or simple carbohydrate modificationor early glycosylation, particularly high mannose modification, and isreduced or not evident in the presence of complex carbohydratemodification.

The specific binding member, which may be an antibody or a fragmentthereof, such as an immunogenic fragment thereof, does not bind to orrecognize normal or wild type cells containing normal or wild type EGFRepitope in the absence of aberrant expression and in the presence ofnormal EGFR post-translational modification. More particularly, thespecific binding member of the invention, may be an antibody or fragmentthereof, which recognizes an EGFR epitope which is present in cellsoverexpressing EGFR (e.g., EGFR gene is amplified) or expressing thede2-7 EGFR, particularly in the presence of aberrant post-translationalmodification, and that is not detectable in cells expressing EGFR undernormal conditions, particularly in the presence of normalpost-translational modification.

The present inventors have discovered novel monoclonal antibodies,exemplified herein by the antibody designated mAb 806, whichspecifically recognize aberrantly expressed EGFR. In particular, theantibodies of the present invention recognize an EGFR epitope which isfound in tumorigenic, hyperproliferative or abnormal cells and is notdetectable in normal or wild type cells, wherein the epitope is enhancedor evident upon aberrant post-translational modification. The antibodiesof the present invention are further exemplified by the antibodies mAb124 and mAb 1133 described herein. The novel antibodies of the inventionalso recognize amplified wild type EGFR and the de2-7 EGFR, yet bind toan epitope distinct from the unique junctional peptide of the de2-7 EGFRmutation. The antibodies of the present invention specifically recognizeaberrantly expressed EGFR, including amplified EGFR and mutant EGFR(exemplified herein by the de2-7 mutation), particularly upon aberrantpost-translational modification. Additionally, while mAb 806 does notrecognize the EGFR when expressed on the cell surface of a glioma cellline expressing normal amounts of EGFR, it does bind to theextracellular domain of the EGFR (sEGFR) immobilized on the surface ofELISA plates, indicating the recognition of a conformational epitope.MAb 806 binds to the surface of A431 cells, which have an amplificationof the EGFR gene but do not express the de2-7 EGFR. Importantly, mAb 806did not bind significantly to normal tissues such as liver and skin,which express levels of endogenous, wild type (wt) EGFR that are higherthan in most other normal tissues, but wherein EGFR is not aberrantlyexpressed or amplified.

The antibodies of the present invention can specifically categorize thenature of EGFR tumors or tumorigenic cells, by staining or otherwiserecognizing those tumors or cells wherein aberrant EGFR expression,including EGFR amplification and/or EGFR mutation, particularlyde2-7EGFR, is present. Further, the antibodies of the present invention,as exemplified by mAb 806, demonstrate significant in vivo anti-tumoractivity against tumors containing amplified EGFR and against de2-7 EGFRpositive xenografts.

The unique specificity of mAb 806, whereby mAb 806 binds to the de2-7EGFR and amplified EGFR but not the normal, wild type EGFR, providesdiagnostic and therapeutic uses to identify, characterize and target anumber of tumor types, for example, head and neck, breast, or prostatetumors and glioma, without the problems associated with normal tissueuptake that may be seen with previously known EGFR antibodies.

Accordingly, the invention provides specific binding proteins, such asantibodies, which bind to the de2-7 EGFR at an epitope which is distinctfrom the junctional peptide but which do not bind to EGFR on normalcells in the absence of amplification of the EGFR gene. Byamplification, it is meant to include that the cell comprises multiplecopies of the EGFR gene.

Preferably the epitope recognized by mAb 806 is located within theregion comprising residues 273-501 of the mature normal or wild typeEGFR sequence. Therefore, also provided are specific binding proteins,such as antibodies, which bind to the de2-7 EGFR at an epitope locatedwithin the region comprising residues 273-501 of the EGFR sequence. Theepitope may be determined by any conventional epitope mapping techniquesknown to the person skilled in the art. Alternatively, the DNA sequenceencoding residues 273-501 could be digested, and the resultant fragmentsexpressed in a suitable host. Antibody binding could be determined asmentioned above.

In a preferred aspect, the antibody is one which has the characteristicsof the antibody which the inventors have identified and characterized,in particular recognizing aberrantly expressed EGFR, as found inamplified EGFR and de2-7EGFR. In a particularly preferred aspect theantibody is the mAb 806, or active fragments thereof. In a furtherpreferred aspect the antibody of the present invention comprises the VHand VL amino acid sequences depicted in FIG. 14 (SEQ ID NO:2) and FIG.15 (SEQ ID NO:4) respectively.

In another aspect, the invention provides an antibody capable ofcompeting with the 806 antibody, under conditions in which at least 10%of an antibody having the VH and VL sequences of the 806 antibody isblocked from binding to de2-7EGFR by competition with such an antibodyin an ELISA assay. In particular, anti-idiotype antibodies arecontemplated and are exemplified herein. The anti-idiotype antibodiesLMH-11, LMH-12 and LMH-13 are provided herein.

An isolated polypeptide consisting essentially of the epitope comprisingresidues 273-501 of the mature normal, wild type EGFR (residues 6-234 ofmature de2-7 EGFR) forms another aspect of the present invention. Thepeptide of the invention is particularly useful in diagnostic assays orkits and therapeutically or prophylactically, including as a tumor oranti-cancer vaccine. Thus compositions of the peptide of the presentinvention include pharmaceutical compositions and immunogeniccompositions.

The binding of an antibody to its target antigen is mediated through thecomplementarity-determining regions (CDRs) of its heavy and lightchains, with the role of CDR3 being of particular importance.Accordingly, specific binding members based on the CDR3 regions of theheavy or light chain, and preferably both, of mAb806 will be usefulspecific binding members for in vivo therapy. The CDRs of the mAb 806antibody are shown in FIGS. 16 and 17.

Accordingly, specific binding proteins such as antibodies which arebased on the CDRs of the mAb 806 antibody identified, particularly theCDR 3 regions, will be useful for targeting tumors with amplified EGFRregardless of their de2-7 EGFR status. As mAb 806 does not bindsignificantly to normal, wild type receptor, there would be nosignificant uptake in normal tissue, a limitation of EGFR antibodiescurrently being developed (18, 19).

In the accompanying drawings, the nucleic acid sequence (SEQ ID NO:1)and translation (SEQ ID NO:2) thereof of the 806 VH gene is shown inFIG. 14. The VL gene of the 806 antibody is shown as FIG. 15 as nucleicacid sequence (SEQ ID NO:3) and predicted amino acid sequence (SEQ IDNO:4). In FIGS. 16 and 17, depicting the VH and VL polypeptide sequencesof mAb 806, the CDRs are indicated in boxes.

In a further aspect, the present invention provides an isolated specificbinding member capable of binding an antigen, wherein said specificbinding member comprises a polypeptide binding domain comprising anamino acid sequence substantially as set out as residues 93-102 of SEQID NO:2. The invention further provides said isolated specific bindingmember which further comprises one or both of the polypeptide bindingdomains substantially as set out as residues 26-35A and 49-64 of SEQ IDNO:2, preferably both. One example of such an embodiment is the sequencesubstantially as shown in SEQ ID NO:2. In a preferred embodiment, thebinding domains are carried by a human antibody framework.

In another aspect, the invention provides an isolated specific bindingmember capable of binding a tumor antigen, wherein said specific bindingmember comprises a polypeptide binding domain comprising a heavy chainsequence comprising at least the CDR3 sequence of SEQ ID NO:2, togetherwith a light chain comprising CDRs whose amino acid sequences aresubstantially as found within SEQ ID NO:4. One example of such anembodiment is the sequence substantially as shown in SEQ ID NO:4. In apreferred embodiment, the CDRs are carried by a human antibodyframework.

In further aspects, the invention provides an isolated nucleic acidwhich comprises a sequence encoding a specific binding member as definedabove, and methods of preparing specific binding members of theinvention which comprise expressing said nucleic acids under conditionsto bring about expression of said binding member, and recovering thebinding member.

Yet a further aspect of the invention are compositions of such bindingproteins with additional binding proteins, such as binding proteinswhich bind to EGFR, preferably inhibiting ligand binding thereto. Suchcompositions can be “one pot” cocktails, kits, and so forth, preferablyformulated for ease of administration.

Specific binding members according to the invention may be used in amethod of treatment or diagnosis of the human or animal body, such as amethod of treatment of a tumor in a human patient which comprisesadministering to said patient an effective amount of a specific bindingmember of the invention.

The present invention also relates to a recombinant DNA molecule orcloned gene, or a degenerate variant thereof, which encodes an antibodyof the present invention; preferably a nucleic acid molecule, inparticular a recombinant DNA molecule or cloned gene, encoding theantibody VH which has a nucleotide sequence or is complementary to a DNAsequence shown in FIG. 14 (SEQ ID NO:1). In another embodiment, thepresent invention also relates to a recombinant DNA molecule or clonedgene, or a degenerate variant thereof, preferably a nucleic acidmolecule, in particular a recombinant DNA molecule or cloned gene,encoding the antibody VL which has a nucleotide sequence or iscomplementary to a DNA sequence shown in FIG. 15 (SEQ ID NO:3).

The present invention also includes polypeptides or antibodies havingthe activities noted herein, and that display the amino acid sequencesset forth and described above and in FIGS. 14 and 15 hereof and selectedfrom SEQ ID NO:2 and 4.

In a further embodiment of the invention, the full DNA sequence of therecombinant DNA molecule or cloned gene provided herein may beoperatively linked to an expression control sequence which may beintroduced into an appropriate host. The invention accordingly extendsto unicellular hosts transformed with the cloned gene or recombinant DNAmolecule comprising a DNA sequence encoding the present VH and/or VL, orportions thereof, of the antibody, and more particularly, the VH and/orVL set forth above and in SEQ ID NO:1 and 3.

The present invention naturally contemplates several means forpreparation of the antibodies and active fragments thereof, including asillustrated herein known recombinant techniques, and the invention isaccordingly intended to cover such synthetic or chimeric antibodypreparations within its scope. The isolation of the cDNA and amino acidsequences disclosed herein facilitates the reproduction of the antibodyof the present invention by such recombinant techniques, andaccordingly, the invention extends to expression vectors prepared fromthe disclosed DNA sequences for expression in host systems byrecombinant DNA techniques, and to the resulting transformed hosts.

The present invention provides drugs or other entities, includingantibodies such as anti-idiotype antibodies, that are capable of bindingto the antibody thereby modulating, inhibiting or potentiating theantibody activity. Thus, anti-idiotype antibodies to mAb806 are providedand exemplified herein. Such anti-idiotype antibodies would be useful inthe development of drugs that would specifically bind the antibodiessuch as mAb806 or its epitope or that would potentiate its activity.

The diagnostic utility of the present invention extends to the use ofthe antibodies of the present invention in assays to characterize tumorsor cellular samples or to screen for tumors or cancer, including invitro and in vivo diagnostic assays.

In an immunoassay, a control quantity of the antibodies, or the like maybe prepared and labeled with an enzyme, a specific binding partnerand/or a radioactive element, and may then be introduced into a cellularsample. After the labeled material or its binding partner(s) has had anopportunity to react with sites within the sample, the resulting massmay be examined by known techniques, which may vary with the nature ofthe label attached.

Specific binding members of the invention may carry a detectable orfunctional label. The specific binding members may carry a radioactivelabel, such as the isotopes ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co,⁵⁹Fe, ⁹⁰Y, ¹²¹I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹¹¹In, ²¹¹At, ¹⁹⁸Au, ⁶⁷Cu, ²²⁵Ac,²¹³Bi, ⁹⁹Tc and ¹⁸⁶Re. When radioactive labels are used, known currentlyavailable counting procedures may be utilized to identify and quantitatethe specific binding members. In the instance where the label is anenzyme, detection may be accomplished by any of the presently utilizedcolorimetric, spectrophotometric, fluorospectrophotometric, amperometricor gasometric techniques known in the art.

The radiolabelled specific binding members, particularly antibodies andfragments thereof, are useful in in vitro diagnostics techniques and inin vivo radioimaging techniques. In a further aspect of the invention,radiolabelled specific binding members, particularly antibodies andfragments thereof, particularly radioimmunoconjugates, are useful inradioimmunotherapy, particularly as radiolabelled antibodies for cancertherapy. In a still further aspect, the radiolabelled specific bindingmembers, particularly antibodies and fragments thereof, are useful inradioimmuno-guided surgery techniques, wherein they can identify andindicate the presence and/or location of cancer cells, precancerouscells, tumor cells, and hyperproliferative cells, prior to, during orfollowing surgery to remove such cells.

Immunoconjugates or antibody fusion proteins of the present invention,wherein the specific binding members, particularly antibodies andfragments thereof, of the present invention are conjugated or attachedto other molecules or agents further include, but are not limited tobinding members conjugated to a chemical ablation agent, toxin,immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent ordrug.

The present invention includes an assay system which may be prepared inthe form of a test kit for the quantitative analysis of the extent ofthe presence of, for instance, amplified EGFR or de2-7EGFR. The systemor test kit may comprise a labeled component prepared by one of theradioactive and/or enzymatic techniques discussed herein, coupling alabel to the antibody, and one or more additional immunochemicalreagents, at least one of which is a free or immobilized components tobe determined or their binding partner(s).

In a further embodiment, the present invention relates to certaintherapeutic methods which would be based upon the activity of thebinding member, antibody, or active fragments thereof, or upon agents orother drugs determined to possess the same activity. A first therapeuticmethod is associated with the prevention or treatment of cancer,including but not limited to head and neck, breast, prostate and glioma.

In particular, the binding members and antibodies of the presentinvention, and in a particular embodiment the 806 antibody whosesequences are presented in SEQ ID NOS: 2 and 4 herein, or activefragments thereof, and chimeric (bispecific) or synthetic antibodiesderived therefrom can be prepared in pharmaceutical compositions,including a suitable vehicle, carrier or diluent, for administration ininstances wherein therapy is appropriate, such as to treat cancer. Suchpharmaceutical compositions may also include methods of modulating thehalf-life of the binding members, antibodies or fragments by methodsknown in the art such as pegylation. Such pharmaceutical compositionsmay further comprise additional antibodies or therapeutic agents.

Thus, a composition of the present invention may be administered aloneor in combination with other treatments, therapeutics or agents, eithersimultaneously or sequentially dependent upon the condition to betreated. In addition, the present invention contemplates and includescompositions comprising the binding member, particularly antibody orfragment thereof, herein described and other agents or therapeutics suchas anti-cancer agents or therapeutics, anti-EGFR agents or antibodies,or immune modulators. More generally these anti-cancer agents may betyrosine kinase inhibitors or phosphorylation cascade inhibitors,post-translational modulators, cell growth or division inhibitors (e.g.anti-mitotics), PDGFR inhibitors or signal transduction inhibitors.Other treatments or therapeutics may include the administration ofsuitable doses of pain relief drugs such as non-steroidalanti-inflammatory drugs (e.g. aspirin, paracetamol, ibuprofen orketoprofen) or opiates such as morphine, or anti-emetics. Thus, theseagents may be anti-EGFR specific agents, such as AG1478, or may be moregeneral anti-cancer and anti-neoplastic agents, non limiting examplesincluding doxorubicin, carboplatin and cisplatin. In addition, thecomposition may be administered with immune modulators, such asinterleukins, tumor necrosis factor (TNF) or other growth factors,cytokines or hormones such as dexamethasone which stimulate the immuneresponse and reduction or elimination of cancer cells or tumors. Thecomposition may also be administered with, or may include combinationsalong with other anti-EGFR antibodies, including but not limited to theanti-EGFR antibodies 528; 225; SC-03; 108 (ATCC HB9764) U.S. Pat. No.6,217,866; 14E1 (U.S. Pat. No. 5,942,602); DH8.3; L8A4; Y10; HuMAX-EGFr(Genmab/Medarex); ICR62; and ABX-EGF (Abgenix).

The present invention also includes binding members, includingantibodies and fragments thereof, which are covalently attached to orotherwise associated with other molecules or agents. These othermolecules or agents include, but are not limited to, molecules(including antibodies or antibody fragments) with distinct recognitioncharacteristics, toxins, ligands, and chemotherapeutic agents.

Other objects and advantages will become apparent to those skilled inthe art from a review of the ensuing detailed description, whichproceeds with reference to the following illustrative drawings, and theattendant claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-F present the results of flow cytometric analysis of gliomacell lines. U87MG (light gray histograms) and U87MG.Δ2-7 (dark grayhistograms) cells were stained with either an irrelevant IgG2b antibody(open histograms), DH8.3 (specific for de2-7 EGFR), MAb 806 or 528(binds both wild type and de2-7 EGFR) as indicated.

FIGS. 2A-C present the results of ELISA of MAb 806, DH8.3 and 528antibodies. (A) binding of increasing concentrations of MAb 806 (▴)DH8.3 (●) or 528 (▪) antibody to sEGFR coated ELISA plates. (B)inhibition of MAb 806 and 528 binding to sEGFR coated ELISA plates byincreasing concentrations of sEGFR in solution. (C) binding ofincreasing concentrations of DH8.3 to the de2-7 junctional peptideillustrates binding curves for mAb 806 and 528 antibodies to immobilizedwild-type sEGFR.

FIGS. 2D-E graphically present the results of BIAcore™ binding studiesusing C-terminal biotinylated peptide and including a monoclonalantibody of the invention, along with other known antibodies, among themthe L8A4 antibody which recognizes the junction peptide of the de2-7EGFR mutant, and controls.

FIG. 3 depicts the internalization of MAb 806 and the DH8.3 antibody.U87MG.Δ2-7 cells were pre-incubated with MAb 806 (▴) or DH8.3 (●) at 4°C., transferred to 37° C. and internalization determined by FACS. Datarepresents mean internalization at each time point±SE of 3 (DH8.3) or 4(MAb 806) separate experiments.

FIGS. 4A-B illustrate biodistribution (% ID/g tumor) of radiolabeled (a)¹²⁵I-MAb 806 and (b) ¹³¹I-DH8.3 in nude mice bearing U87MG andU87MG.Δ2-7 xenografts. Each point represents the mean of 5 mice±SEexcept for 1 hr where n=4.

FIGS. 5A-B illustrate biodistribution of radiolabeled ¹²⁵I-MAb 806 (openbar) and ¹³¹I-DH8.3 (filled bar) antibodies expressed as (a) tumor:bloodor (b) tumor:liver ratios in nude mice bearing U87MG.Δ2-7 xenografts.Each bar represents the mean of 5 mice±SE except for 1 hr where n=4

FIGS. 6A-C illustrate flow cytometric analysis of cell lines containingamplification of the EGFR gene. A431 cells were stained with either MAb806, DH8.3 or 528 (black histograms) and compared to an irrelevant IgG2bantibody (open histogram)

FIGS. 7A-B illustrate biodistribution (% ID/g tumor) of radiolabeled (a)¹²⁵I-MAb 806 and (b) ¹³¹I-528 in nude mice bearing U87MG.Δ2-7 and A431xenografts.

FIGS. 8A-D illustrate biodistribution of radiolabeled ¹²⁵I-MAb 806 (openbar) and ¹³¹I-528 (filled bar) antibodies expressed as (a,b) tumor:bloodor (c,d) tumor:liver ratios in nude mice bearing (a,c) U87MGA2-7 and(b,d) A431 xenografts.

FIGS. 9A-B illustrate anti-tumor effect of mAb 806 on A) U87MG and B)U87MG.Δ2-7 xenograft growth rates in a preventative model. 3×10⁶ U87MGor U87MG.Δ2-7 cells were injected s.c. into both flanks of 4-6 week oldBALB/c nude mice, (n=5) at day 0. Mice were injected i.p. with either 1mg of mAb 806 (●); 0.1 mg of mAb 806 (▴); or vehicle (∘) starting oneday prior to tumor cell inoculation. Injections were given three timesper week for two weeks as indicated by the arrows. Data are expressed asmean tumor volume±S.E.

FIGS. 10A-C illustrate the anti-tumor effect of mAb 806 on A) U87MG, B)U87MG.Δ2-7 and C) U87MG.wtEGFR xenografts in an established model. 3×10⁶U87MG, U87MG.Δ2-7, or U87MG.wtEGFR cells, were injected s.c. into bothflanks of 4-6 week old BALB/c nude mice, (n=5). Mice were injected i.p.with either 1 mg doses of mAb 806 (●); 0.1 mg doses of mAb 806 (▴); orvehicle (∘) starting when tumors had reached a mean tumor volume of65-80 mm³. Injections were given three times per week for two weeks asindicated by the arrows. Data are expressed as mean tumor volume±S.E.

FIGS. 11A-B illustrate anti-tumor effect of mAb 806 on A431 xenograftsin A) preventative and B) established models. 3×10⁶ A431 cells wereinjected s.c. into both flanks of 4-6 week old BALB/c nude mice (n=5).Mice were injected i.p. with either 1 mg doses of mAb 806 (●); orvehicle (∘), starting one day prior to tumor cell inoculation in thepreventative model, or when tumors had reached a mean tumor volume of200 mm³. Injections were given three times per week for two weeks asindicated by the arrows. Data are expressed as mean tumor volume±S.E.

FIG. 12 illustrates the anti-tumor effect of treatment with mAb 806combined with treatment with AG1478 on A431 xenografts in a preventativemodel. Data are expressed as mean tumor volume±S.E.

FIG. 13 depicts antibody 806 binding to A431 cells in the presence ofincreasing concentrations of AG1478 (0.5 μM and 5 μM).

FIGS. 14A-B illustrate (A) the nucleic acid sequence and (B) the aminoacid translation thereof of the 806 VH gene (SEQ ID NO:1 and SEQ IDNO:2, respectively).

FIGS. 15A-B illustrate (A) the nucleic acid sequence and (B) the aminoacid translation thereof of the 806 VL gene (SEQ ID NO:3 and SEQ IDNO:4, respectively).

FIG. 16 shows the VH sequence (SEQ ID NO:11) numbered according toKabat, with the CDRs boxed. Key residues of the VH are 24, 37, 48, 67and 78.

FIG. 17 shows the VL sequence (SEQ ID NO:12) numbered according toKabat, with the CDRs boxed. Key residues of the VL are 36, 46, 57 and71.

FIGS. 18A-D show the results of in vivo studies designed to determinethe therapeutic effect of combination antibody therapy, particularlymAb806 and the 528 antibody. Mice received inoculations of U87MG.Δ2-7 (Aand B), U87MG.DK (C), or A431 (D) cells.

FIGS. 19A-D show analysis of internalization by electron microscopy.U87MG.Δ2-7 cells were pre-incubated with MAb 806 or DH8.3 followed bygold conjugated anti-mouse IgG at 4° C., transferred to 37° C. andinternalization examined at various time points by electron microscopy.(A) localization of the DH8.3 antibody to a coated pit (arrow) after 5min; (B) internalization of MAb 806 by macropinocytosis (arrow) after 2min; (C) localization of DH8.3 to lysosomes (arrow) after 20 min; (D)localization of MAb 806 to lysosomes (arrow) after 30 min. Originalmagnification for all images is ×30,000.

FIG. 20 shows autoradiography of a U87MG.Δ2-7 xenograft sectioncollected 8 hr after injection of ¹²⁵I-MAb 806.

FIGS. 21A-B show flow cytometric analysis of cell lines containingamplification of the EGFR gene. HN5 and MDA-468 cells were stained withan irrelevant IgG2b antibody (open histogram with dashed line), MAb 806(black histogram) or 528 (open histogram with closed lines). The DH8.3antibody was completely negative on both cell lines (data not shown).

FIG. 22 shows immunoprecipitation of EGFR from cell lines. The EGFR wasimmunoprecipitated from ³⁵ S-labeled U87MG.Δ2-7 or A431 cells with MAb806, sc-03 antibody or a IgG2b isotype control. Arrows at the sideindicate the position of the de2-7 and wt EGFR. Identical bandingpatterns were obtained in 3 independent experiments.

FIG. 23 shows autoradiography of an A431 xenograft section collected 24hr after injection of ¹²⁵I-MAb 806, areas of localization to viabletissue are indicated (arrows).

FIGS. 24A-E illustrate: A and B, extended survival of nude mice bearingintracranial U87MG.ΔEGFR (A) and LN-Z308.ΔEGFR (B) xenografts withsystemic mAb 806 treatment. U87MG.ΔGFR cells (1×10⁵) or LN-Z308.ΔEGFRcells (5×10⁵) were implanted into nude mice brains, and the animals weretreated with either mAb 806, PBS, or isotype IgG from postimplantationdays 0 through 14. C and D, growth inhibition of intracranial tumors bymAb 806 treatment. Nude mice (five per group), treated with either mAb806 or the isotype IgG control, were euthanized on day 9 forU87MG.ΔEGFR(C) and on day 15 for LN-Z308.ΔEGFR (D), and their brainswere harvested, fixed, and sectioned. Data were calculated by taking thetumor volume of control as 100%. Values are mean±SD. ***, P<0.001;control versus mAb 806. Arrowheads, tumor tissue. E, extended survivalof nude mice bearing intracranial U87MG.ΔEGFR xenografts withintratumoral mAb 806 treatment. U87MG.ΔEGFR cells were implanted asdescribed. Ten mg of mAb 806 or isotype IgG control in a volume of 5 μlwere injected at the tumor-injection site every other day starting atday 1 for five times.

FIG. 25A-C mAb 806 extends survival of mice with U87.MG.wtEGFR braintumors but not with U87MG.DK or U87MG brain tumors. U87MG (A), U87MG.DK(B), or U87MG.wtEGFR(C) cells (5×10⁵) were implanted into nude micebrains, and the animals were treated with mAb 806 from postimplantationdays 0 through 14 followed by observation after discontinuation oftherapy.

FIGS. 26A-B illustrate: A, FACS analysis of mAb 806 reactivity withU87MG cell lines. U87MG, U87MGΔEGFR, U87MG.DK, and U87MG.wtEGFR cellswere stained with anti-EGFR mAbs 528, EGFR.1, and anti-ΔEGFR antibody,mAb 806. Monoclonal EGFR.1 antibody recognized wtEGFR exclusively andmonoclonal 528 antibody reacted with both wtEGFR and ΔEGFR. mAb 806reacted intensively with U87MG.ΔEGFR and U87MG.DK and weakly withU87MG.wtEGFR. Bars on the abscissa, maximum staining of cells in theabsence of primary antibody. Results were reproduced in threeindependent experiments. B, mAb 806 immunoprecipitation of EGFR forms.Mutant and wtEGFR were immunoisolated with anti-EGFR antibodies, 528, orEGFR.1, or anti-ΔEGFR antibody, mAb 806, from (Lane 1) U87MG, (Lane 2)U87Δ.EGFR, (Lane 3) U87MG.DK, and (Lane 4) U87MG.wtEGFR cells and werethen detected by Western blotting with anti-pan EGFR antibody, C13.

FIGS. 27A-B illustrate systemic treatment with mAb 806 decreases thephosphorylation of ΔEGFR and Bcl-X_(L) expression in U87MG.ΔEGFR braintumors. U87MG.ΔEGFR tumors were resected at day 9 of mAb 806 treatment,immediately frozen in liquid nitrogen and stored at −80° C. before tumorlysate preparation. A, Western blot analysis of expression and thedegree of autophosphorylation of ΔEGFR. Thirty μg of tumor lysates weresubjected to SDS-polyacrylamide gels, transferred to nitrocellulosemembranes, and probed with anti-phosphotyrosine mAb, then were strippedand re-probed with anti-EGFR antibody, C13. B, Western blotting ofBcl-X_(L) by using the same tumor lysates as in A. Membranes were probedwith anti-human Bcl-X_(L) polyclonal antibody. Lanes 1 and 2,U87MG.ΔEGFR brain tumors treated with isotype control; Lanes 3 and 4,U87MG.ΔEGFR brain tumors treated with mAb 806.

FIG. 28 illustrates that mAb 806 treatment leads to a decrease in growthand vasculogenesis and to increases in apoptosis and accumulatingmacrophages in U87MG.ΔEGFR tumors. Tumor sections were stained forKi-67. Cell proliferative index was assessed by the percentage of totalcells that were Ki-67 positive from four randomly selected high-powerfields (×400) in intracranial tumors from four mice of each group. Dataare the mean±SE. Apoptotic cells were detected by TUNEL assay. Apoptoticindex was assessed by the ratio of TUNEL-positive cells:total number ofcells from four randomly selected high-power fields (×400) inintracranial tumors from four mice of each group. Data are the mean±SE.Tumor sections were immunostained with anti-CD31 antibody. MVAs wereanalyzed by computerized image analysis from four randomly selectedfields (×200) from intracranial tumors from four mice of each group.Peritumoral infiltrates of macrophages in mAb 806-treated U87MG.ΔEGFRtumors. Tumor sections were stained with anti-F4/80 antibody.

FIGS. 29A-F show cytometric analysis of parental and transfected U87MGglioma cell lines. Cells were stained with either an irrelevant IgG2bantibody (open histograms) or the 528 antibody or mAb 806 (filledhistograms) as indicated.

FIG. 30 show immunoprecipitation of EGFR from cell lines. The EGFR wasimmunoprecipitated from ³⁵S-labeled U87MG.wtEGFR, U87MG.Δ2-7, and A431cells with mAb806 (806), sc-03 antibody (c-term), or a IgG2b isotypecontrol (con). Arrows, position of the de2-7 and wt EGFR.

FIG. 31 shows representative H&E-stained paraffin sections of U87MG.Δ2-7and U87MG.wtEGFR xenografts. U87MG.Δ2-7 (collected 24 days after tumorinoculation) and U87MG.wtEGFR (collected 42 days after tumorinoculation) xenografts were excised from mice treated as described inFIGS. 10A-C above, and stained with H&E. Vehicle-treated U87MG.Δ2-7(collected 18 days after tumor inoculation) and U87MG.wtEGFR (collected37 days after tumor inoculation) xenografts showed very few areas ofnecrosis (left panel), whereas extensive necrosis (arrows) was observedin both U87MG.Δ2-7 and U87MG.wtEGFR xenografts treated with mAb 806(right panel).

FIG. 32 shows immunohistochemical analysis of EGFR expression in frozensections derived from U87MG, U87MG.Δ2-7, and U87MG.wtEGFR xenografts.Sections were collected at the time points described in FIG. 31 above.Xenograft sections were immunostained with the 528 antibody (left panel)and mAb 806 (right panel). No decreased immunoreactivity to either wtEGFR, amplified EGFR, or de2-7 EGFR was observed in xenografts treatedwith mAb 806. Consistent with the in vitro data, parental U87MGxenografts were positive for 528 antibody but were negative for mAb 806staining.

FIGS. 33A-B show schematic representations of generated bicistronicexpression constructs. Transcription of the chimeric antibody chains isinitiated by Elongation Factor-1 promoter and terminated by a strongartificial termination sequence. IRES sequences were introduced betweencoding regions of light chain and NeoR and heavy chain and dhfr gene.

FIGS. 34A-B show biodistribution analysis of the ch806 radiolabeled witheither A) ¹²⁵I or B) ¹¹¹In was performed in BALB/c nude mice bearingU87MG-de2-7 xenograft tumors. Mice were injected with 5 ug ofradiolabeled antibody and in groups of 4 mice per time point, sacrificedat either 8, 28, 48 or 74 hours. Organs were collected, weighed andradioactivity measured in a gamma counter.

FIGS. 35A-B depict (A) the % ID gram tumor tissue and (B) the tumor toblood ratio. Indium-111 antibody shows approximately 30% ID/gram tissueand a tumor to blood ratio of 4.0.

FIG. 36 depicts the therapeutic efficacy of chimeric antibody ch806 inan established tumor model. 3×10⁶ U87MG.Δ2-7 cells in 100 μl of PBS wereinoculated s.c. into both flanks of 4-6 week old female nude mice. ThemAb806 was included as a positive control. Treatment was started whentumors had reached a mean volume of 50 mm³ and consisted of 1 mg ofch806 or mAb806 given i.p. for a total of 5 injections on the daysindicated. Data was expressed as mean tumor volume+/−S.E. for eachtreatment group.

FIGS. 37A-B show CDC Activity on Target A) U87MG.de2-7 and B) A431 cellsfor anti-EGFR chimeric IgG1 antibodies ch806 and control cG250. Mean(bars; ±SD) percent cytotoxicity of triplicate determinations arepresented.

FIGS. 38A-B show ADCC on target A) U87MG.de2-7 and B) A431 cells atEffector:Target cell ratio of 50:1 mediated by ch806 and isotype controlcG250 (0-10 ug/ml). Results are expressed as mean (bars; ±SD) percentcytotoxicity of triplicate determinations.

FIG. 39 shows ADCC mediated by 1 μg/ml parental mAb 806 and ch806 ontarget U87MG.de2-7 cells over a range of Effector:Target ratios. Mean(bars; ±SD) of triplicate determinations are presented.

FIGS. 40A-B illustrate that twenty-five hybridomas producing antibodiesthat bound ch806 but not huIgG were initially selected. Four of theseanti-ch806 hybridomas with high affinity binding (clones 3E3, 5B8, 9D6and 4D8) were subsequently pursued for clonal expansion from singlecells by limiting dilution and designated Ludwig Institute for CancerResearch Melbourne Hybridoma (LMH)-11, -12, -13 and -14, respectively.In addition, two hybridomas that produced mAbs specific for huIgG werealso cloned and characterized further: clones 2C10 (LMH-15) and 2B8(LMH-16).

FIGS. 41A-C illustrate that after clonal expansion, the hybridomaculture supernatants were examined in triplicate by ELISA for theability to neutralize ch806 or mAb 806 antigen binding activity withsEGFR621. Mean (±SD) results demonstrated the antagonist activity ofanti-idiotype mAbs LMH-11, -12, -13 and -14 with the blocking insolution of both ch806 and murine mAb 806 binding to plates coated withsEGFR (LMH-14 not shown).

FIGS. 42A-C illustrate that microtitre plates were coated with 10 μg/mlpurified A) LMH-11, B) LMH-12 and C) LMH-13. The three purified cloneswere compared for their ability to capture ch806 or mAb 806 in sera or1% FCS/Media and then detect bound ch806 or mAb806. Isotype controlantibodies hu3S193 and m3S193 in serum and 1% FCS/Media were included inaddition to controls for secondary conjugate avidin-HRP and ABTSsubstrate. Results are presented as mean (±SD) of triplicate samplesusing biotinylated-LMH-12 (10

g/ml) for detection and indicate LMH-12 used for capture and detectionhad the highest sensitivity for ch806 in serum (3 ng/ml) with negligiblebackground binding.

FIG. 43 illustrates validation of the optimal pharmacokinetic ELISAconditions using 1 μg/ml anti-idiotype LMH-12 and 1 μg/ml biotinylatedLMH-12 for capture and detection, respectively. Three separate ELISAswere performed in quadruplicate to measure ch806 in donor serum (●) fromthree healthy donors or 1% BSA/media (▪) with isotype control hu3S193 inserum (σ) or 1% BSA/media (τ). Controls for secondary conjugateavidin-HRP (υ) and ABTS substrate (hexagon) alone were also includedwith each ELISA. Mean (±SD) results demonstrate highly reproduciblebinding curves for measuring ch806 (2 μg/ml-1.6 ng/ml) in sera with a 3ng/ml limit of detection. (n=12; 1-100 ng/ml, Coefficient of Variation<25%; 100 ng/ml-5 μg/ml, Coefficient of Variation <15%). No backgroundbinding was evident with any of the three sera tested and negligiblebinding was observed with isotype control hu3S193.

FIG. 44 depicts an immunoblot of recombinant sEGFR expressed in CHOcells, blotted with mAb806. Recombinant sEGFR was treated with PNGaseFto remove N-linked glycosylation (deglycosylated), or untreated(untreated), the protein was run on SDS-PAGE, transferred to membraneand immunoblotted with mAb 806.

FIG. 45 depicts immunoprecipitation of EGFR from ³⁵S-labelled cell lines(U87MG Δ2-7, U87MG-wtEGFR, and A431) with different antibodies (SC-03,806 and 528 antibodies).

FIG. 46 depicts immunoprecipitation of EGFR from different cells (A431and U87MGΔ2-7) at different time points (time 0 to 240 minutes) afterpulse-labelling with ³⁵S methionine/cysteine. Antibodies 528 and 806 areused for immunoprecipitation.

FIG. 47 depicts immunoprecipitation of EGFR from various cell lines(U87MGΔ2-7, U87MG-wtEGFR and A431) with various antibodies (SC-03, 806and 528) in the absence of (−) and after Endo H digestion (+) to removehigh mannose type carbohydrates.

FIG. 48 depicts cell surface iodination of the A431 and U87MGΔ2-7 celllines followed by immunoprecipitation with the 806 antibody, and with orwithout Endo H digestion, confirming that the EGFR bound by mAb 806 onthe cell surface of A431 cells is an EndoH sensitive form.

DETAILED DESCRIPTION

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R.I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

A. Terminology

The term “specific binding member” describes a member of a pair ofmolecules which have binding specificity for one another. The members ofa specific binding pair may be naturally derived or wholly or partiallysynthetically produced. One member of the pair of molecules has an areaon its surface, or a cavity, which specifically binds to and istherefore complementary to a particular spatial and polar organisationof the other member of the pair of molecules. Thus the members of thepair have the property of binding specifically to each other. Examplesof types of specific binding pairs are antigen-antibody, biotin-avidin,hormone-hormone receptor, receptor-ligand, enzyme-substrate. Thisapplication is concerned with antigen-antibody type reactions.

The term “aberrant expression” in its various grammatical forms may meanand include any heightened or altered expression or overexpression of aprotein in a tissue, e.g. an increase in the amount of a protein, causedby any means including enhanced expression or translation, modulation ofthe promoter or a regulator of the protein, amplification of a gene fora protein, or enhanced half-life or stability, such that more of theprotein exists or can be detected at any one time, in contrast to anon-overexpressed state. Aberrant expression includes and contemplatesany scenario or alteration wherein the protein expression orpost-translational modification machinery in a cell is taxed orotherwise disrupted due to enhanced expression or increased levels oramounts of a protein, including wherein an altered protein, as inmutated protein or variant due to sequence alteration, deletion orinsertion, or altered folding is expressed.

It is important to appreciate that the term “aberrant expression” hasbeen specifically chosen herein to encompass the state where abnormal(usually increased) quantities/levels of the protein are present,irrespective of the efficient cause of that abnormal quantity or level.Thus, abnormal quantities of protein may result from overexpression ofthe protein in the absence of gene amplification, which is the case e.g.in many cellular/tissue samples taken from the head and neck of subjectswith cancer, while other samples exhibit abnormal protein levelsattributable to gene amplification.

In this latter connection, certain of the work of the inventors that ispresented herein to illustrate the invention includes the analysis ofsamples certain of which exhibit abnormal protein levels resulting fromamplification of EFGR. This therefore accounts for the presentationherein of experimental findings where reference is made to amplificationand for the use of the terms “amplification/amplified” and the like indescribing abnormal levels of EFGR. However, it is the observation ofabnormal quantities or levels of the protein that defines theenvironment or circumstance where clinical intervention as by resort tothe binding members of the invention is contemplated, and for thisreason, the present specification considers that the term “aberrantexpression” more broadly captures the causal environment that yields thecorresponding abnormality in EFGR levels.

Accordingly, while the terms “overexpression” and “amplification” intheir various grammatical forms are understood to have distincttechnical meanings, they are to be considered equivalent to each other,insofar as they represent the state where abnormal EFGR protein levelsare present in the context of the present invention. Consequently, theterm “aberrant expression” has been chosen as it is believed to subsumethe terms “overexpression” and “amplification” within its scope for thepurposes herein, so that all terms may be considered equivalent to eachother as used herein.

The term “antibody” describes an immunoglobulin whether natural orpartly or wholly synthetically produced. The term also covers anypolypeptide or protein having a binding domain which is, or ishomologous to, an antibody binding domain. CDR grafted antibodies arealso contemplated by this term.

As antibodies can be modified in a number of ways, the term “antibody”should be construed as covering any specific binding member or substancehaving a binding domain with the required specificity. Thus, this termcovers antibody fragments, derivatives, functional equivalents andhomologues of antibodies, including any polypeptide comprising animmunoglobulin binding domain, whether natural or wholly or partiallysynthetic. Chimeric molecules comprising an immunoglobulin bindingdomain, or equivalent, fused to another polypeptide are thereforeincluded. Cloning and expression of chimeric antibodies are described inEP-A-0120694 and EP-A-0125023 and U.S. Pat. Nos. 4,816,397 and4,816,567.

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward, E. S. et al., Nature 341, 544-546 (1989)) which consistsof a VH domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, abivalent fragment comprising two linked Fab fragments (vii) single chainFv molecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston etal, PNAS USA, 85, 5879-5883, 1988); (viii) multivalent antibodyfragments (scFv dimers, trimers and/or tetramers (Power and Hudson, J.Immunol. Methods 242: 193-204 9 (2000))(ix) bispecific single chain Fvdimers (PCT/US92/09965) and (x) “diabodies”, multivalent ormultispecific fragments constructed by gene fusion (WO94/13804; P.Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, (1993)).

An “antibody combining site” is that structural portion of an antibodymolecule comprised of light chain or heavy and light chain variable andhypervariable regions that specifically binds antigen.

The phrase “antibody molecule” in its various grammatical forms as usedherein contemplates both an intact immunoglobulin molecule and animmunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contains the paratope, including thoseportions known in the art as Fab, Fab′, F(ab′)₂ and F(v), which portionsare preferred for use in the therapeutic methods described herein.

Antibodies may also be bispecific, wherein one binding domain of theantibody is a specific binding member of the invention, and the otherbinding domain has a different specificity, e.g. to recruit an effectorfunction or the like. Bispecific antibodies of the present inventioninclude wherein one binding domain of the antibody is a specific bindingmember of the present invention, including a fragment thereof, and theother binding domain is a distinct antibody or fragment thereof,including that of a distinct anti-EGFR antibody, for instance antibody528 (U.S. Pat. No. 4,943,533), the chimeric and humanized 225 antibody(U.S. Pat. No. 4,943,533 and WO/9640210), an anti-de2-7 antibody such asDH8.3 (Hills, D. et al (1995) Int. J. Cancer 63(4):537-543), antibodyL8A4 and Y10 (Reist, C J et al (1995) Cancer Res. 55(19):4375-4382;Foulon C F et al. (2000) Cancer Res. 60(16):4453-4460), ICR62(Modjtahedi H et al (1993) Cell Biophys. January-June; 22(1-3):129-46;or the antibody of Wikstrand et al (Wikstrand C. et al (1995) CancerRes. 55(14):3140-3148). The other binding domain may be an antibody thatrecognizes or targets a particular cell type, as in a neural or glialcell-specific antibody. In the bispecific antibodies of the presentinvention the one binding domain of the antibody of the invention may becombined with other binding domains or molecules which recognizeparticular cell receptors and/or modulate cells in a particular fashion,as for instance an immune modulator (e.g., interleukin(s)), a growthmodulator or cytokine (e.g. tumor necrosis factor (TNF), andparticularly, the TNF bispecific modality demonstrated in U.S. Ser. No.60/355,838 filed Feb. 13, 2002 incorporated herein in its entirety) or atoxin (e.g., ricin) or anti-mitotic or apoptotic agent or factor.

Fab and F(ab′)₂ portions of antibody molecules may be prepared by theproteolytic reaction of papain and pepsin, respectively, onsubstantially intact antibody molecules by methods that are well-known.See for example, U.S. Pat. No. 4,342,566 to Theofilopolous et al. Fab′antibody molecule portions are also well-known and are produced fromF(ab′)₂ portions followed by reduction of the disulfide bonds linkingthe two heavy chain portions as with mercaptoethanol, and followed byalkylation of the resulting protein mercaptan with a reagent such asiodoacetamide. An antibody containing intact antibody molecules ispreferred herein.

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may alsocontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bispecific(chimeric) monoclonal antibody.

The term “antigen binding domain” describes the part of an antibodywhich comprises the area which specifically binds to and iscomplementary to part or all of an antigen. Where an antigen is large,an antibody may bind to a particular part of the antigen only, whichpart is termed an epitope. An antigen binding domain may be provided byone or more antibody variable domains. Preferably, an antigen bindingdomain comprises an antibody light chain variable region (VL) and anantibody heavy chain variable region (VH).

“Post-translational modification” may encompass any one of orcombination of modification(s), including covalent modification, which aprotein undergoes after translation is complete and after being releasedfrom the ribosome or on the nascent polypeptide cotranslationally.Post-translational modification includes but is not limited tophosphorylation, myristylation, ubiquitination, glycosylation, coenzymeattachment, methylation and acetylation. Post-translational modificationcan modulate or influence the activity of a protein, its intracellularor extracellular destination, its stability or half-life, and/or itsrecognition by ligands, receptors or other proteins Post-translationalmodification can occur in cell organelles, in the nucleus or cytoplasmor extracellularly.

The term “specific” may be used to refer to the situation in which onemember of a specific binding pair will not show any significant bindingto molecules other than its specific binding partner(s). The term isalso applicable where e.g. an antigen binding domain is specific for aparticular epitope which is carried by a number of antigens, in whichcase the specific binding member carrying the antigen binding domainwill be able to bind to the various antigens carrying the epitope.

The term “comprise” generally used in the sense of include, that is tosay permitting the presence of one or more features or components.

The term “consisting essentially of” refers to a product, particularly apeptide sequence, of a defined number of residues which is notcovalently attached to a larger product. In the case of the peptide ofthe invention referred to above, those of skill in the art willappreciate that minor modifications to the N- or C-terminal of thepeptide may however be contemplated, such as the chemical modificationof the terminal to add a protecting group or the like, e.g. theamidation of the C-terminus.

The term “isolated” refers to the state in which specific bindingmembers of the invention, or nucleic acid encoding such binding memberswill be, in accordance with the present invention. Members and nucleicacid will be free or substantially free of material with which they arenaturally associated such as other polypeptides or nucleic acids withwhich they are found in their natural environment, or the environment inwhich they are prepared (e.g. cell culture) when such preparation is byrecombinant DNA technology practised in vitro or in vivo. Members andnucleic acid may be formulated with diluents or adjuvants and still forpractical purposes be isolated—for example the members will normally bemixed with gelatin or other carriers if used to coat microtitre platesfor use in immunoassays, or will be mixed with pharmaceuticallyacceptable carriers or diluents when used in diagnosis or therapy.Specific binding members may be glycosylated, either naturally or bysystems of heterologous eukaryotic cells, or they may be (for example ifproduced by expression in a prokaryotic cell) unglycosylated.

Also, as used herein, the terms “glycosylation” and “glycosylated”includes and encompasses the post-translational modification ofproteins, termed glycoproteins, by addition of oligosaccharides.Oligosaccharides are added at glycosylation sites in glycoproteins,particularly including N-linked oligosaccharides and O-linkedoligosaccharides. N-linked oligosaccharides are added to an Asn residue,particularly wherein the Asn residue is in the sequence N—X—S/T, where Xcannot be Pro or Asp, and are the most common ones found inglycoproteins. In the biosynthesis of N-linked glycoproteins, a highmannose type oligosaccharide (generally comprised of dolichol,N-Acetylglucosamine, mannose and glucose is first formed in theendoplasmic reticulum (ER). The high mannose type glycoproteins are thentransported from the ER to the Golgi, where further processing andmodification of the oligosaccharides occurs. O-linked oligosaccharidesare added to the hydroxyl group of Ser or Thr residues. In O-linkedoligosaccharides, N-Acetylglucosamine is first transferred to the Ser orThr residue by N-Acetylglucosaminyltransferase in the ER. The proteinthen moves to the Golgi where further modification and chain elongationoccurs. O-linked modifications can occur with the simple addition of theOGlcNAc monosaccharide alone at those Ser or Thr sites which can alsounder different conditions be phosphorylated rather than glycosylated.

As used herein, “pg” means picogram, “ng” means nanogram, “ug” or “μg”mean microgram, “mg” means milligram, “ul” or “μl” mean microliter, “ml”means milliliter, “l” means liter.

The terms “806 antibody”, “mAb806”, “ch806” and any variants notspecifically listed, may be used herein interchangeably, and as usedthroughout the present application and claims refer to proteinaceousmaterial including single or multiple proteins, and extends to thoseproteins having the amino acid sequence data described herein andpresented in SEQ ID NO:2 and SEQ ID NO:4, and the chimeric antibodych806 which is incorporated in and forms a part of SEQ ID NOS: 7 and 8,and the profile of activities set forth herein and in the Claims.Accordingly, proteins displaying substantially equivalent or alteredactivity are likewise contemplated. These modifications may bedeliberate, for example, such as modifications obtained throughsite-directed mutagenesis, or may be accidental, such as those obtainedthrough mutations in hosts that are producers of the complex or itsnamed subunits. Also, the terms “806 antibody”, “mAb806” and “ch806” areintended to include within their scope proteins specifically recitedherein as well as all substantially homologous analogs and allelicvariations.

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfuctional property of immunoglobulin-binding is retained by thepolypeptide. NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxy group presentat the carboxy terminus of a polypeptide. In keeping with standardpolypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969),abbreviations for amino acid residues are shown in the following Tableof Correspondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid W Trp tryptophan R Arg arginine D Asp aspartic acid NAsn asparagine C Cys cysteine

It should be noted that all amino-acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues. The above Table ispresented to correlate the three-letter and one-letter notations whichmay appear alternately herein.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single strandedform, or a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular double-stranded DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined by mapping with nuclease S1), as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequencesin addition to the -10 and -35 consensus sequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

The term “oligonucleotide,” as used herein in referring to the probe ofthe present invention, is defined as a molecule comprised of two or moreribonucleotides, preferably more than three. Its exact size will dependupon many factors which, in turn, depend upon the ultimate function anduse of the oligonucleotide.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand, isinduced, i.e., in the presence of nucleotides and an inducing agent suchas a DNA polymerase and at a suitable temperature and pH. The primer maybe either single-stranded or double-stranded and must be sufficientlylong to prime the synthesis of the desired extension product in thepresence of the inducing agent. The exact length of the primer willdepend upon many factors, including temperature, source of primer anduse of the method. For example, for diagnostic applications, dependingon the complexity of the target sequence, the oligonucleotide primertypically contains 15-25 or more nucleotides, although it may containfewer nucleotides.

The primers herein are selected to be “substantially” complementary todifferent strands of a particular target DNA sequence. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being complementary to the strand.Alternatively, non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence of the strand to hybridizetherewith and thereby form the template for the synthesis of theextension product.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

It should be appreciated that also within the scope of the presentinvention are DNA sequences encoding specific binding members(antibodies) of the invention which code for e.g. an antibody having thesame amino acid sequence as SEQ ID NO:2 or SEQ ID NO:4, but which aredegenerate to SEQ ID NO:2 or SEQ ID NO:4. By “degenerate to” is meantthat a different three-letter codon is used to specify a particularamino acid. It is well known in the art that the following codons can beused interchangeably to code for each specific amino acid:

Phenylalanine (Phe or F) UUU or UUC Leucine (Leu or L) UUA or UUG or CUUor CUC or CUA or CUG Isoleucine (Ile or I) AUU or AUC or AUA Methionine(Met or M) AUG Valine (Val or V) GUU or GUC of GUA or GUG Serine (Ser orS) UCU or UCC or UCA or UCG or AGU or AGC Proline (Pro or P) CCU or CCCor CCA or CCG Threonine (Thr or T) ACU or ACC or ACA or ACG Alanine (Alaor A) GCU or GCG or GCA or GCG Tyrosine (Tyr or Y) UAU or UAC Histidine(His or H) CAU or CAC Glutamine (Gln or Q) CAA or CAG Asparagine (Asn orN) AAU or AAC Lysine (Lys or K) AAA or AAG Aspartic Acid (Asp or D) GAUor GAC Glutamic Acid (Glu or E) GAA or GAG Cysteine (Cys or C) UGU orUGC Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG Glycine(Gly or G) GGU or GGC or GGA or GGG Tryptophan (Trp or W) UGGTermination codon UAA (ochre) or UAG (amber) or UGA (opal)

It should be understood that the codons specified above are for RNAsequences. The corresponding codons for DNA have a T substituted for U.

Mutations can be made in SEQ ID NO:2 or SEQ ID NO:4 such that aparticular codon is changed to a codon which codes for a different aminoacid. Such a mutation is generally made by making the fewest nucleotidechanges possible. A substitution mutation of this sort can be made tochange an amino acid in the resulting protein in a non-conservativemanner (i.e., by changing the codon from an amino acid belonging to agrouping of amino acids having a particular size or characteristic to anamino acid belonging to another grouping) or in a conservative manner(i.e., by changing the codon from an amino acid belonging to a groupingof amino acids having a particular size or characteristic to an aminoacid belonging to the same grouping). Such a conservative changegenerally leads to less change in the structure and function of theresulting protein. A non-conservative change is more likely to alter thestructure, activity or function of the resulting protein. The presentinvention should be considered to include sequences containingconservative changes which do not significantly alter the activity orbinding characteristics of the resulting protein.

The following is one example of various groupings of amino acids:

Amino Acids with Nonpolar R Groups

Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine,Tryptophan, Methionine

Amino Acids with Uncharged Polar R Groups

Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine

Amino Acids with Charged Polar R Groups (Negatively Charged at pH 6.0)

Aspartic acid, Glutamic acid

Basic Amino Acids (Positively Charged at pH 6.0)

Lysine, Arginine, Histidine (at pH 6.0)

Another Grouping May be Those Amino Acids with Phenyl Groups:

Phenylalanine, Tryptophan, Tyrosine

Another Grouping May be According to Molecular Weight (i.e., Size of RGroups):

Glycine 75 Alanine 89 Serine 105 Proline 115 Valine 117 Threonine 119Cysteine 121 Leucine 131 Isoleucine 131 Asparagine 132 Aspartic acid 133Glutamine 146 Lysine 146 Glutamic acid 147 Methionine 149 Histidine (atpH 6.0) 155 Phenylalanine 165 Arginine 174 Tyrosine 181 Tryptophan 204

Particularly preferred substitutions are:

-   -   Lys for Arg and vice versa such that a positive charge may be        maintained;    -   Glu for Asp and vice versa such that a negative charge may be        maintained;    -   Ser for Thr such that a free —OH can be maintained; and    -   Gln for Asn such that a free NH₂ can be maintained.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced a potential site for disulfide bridges with another Cys. A His may be introduced as a particularly “catalytic” site (i.e., H is canact as an acid or base and is the most common amino acid in biochemicalcatalysis). Pro may be introduced because of its particularly planarstructure, which induces β-turns in the protein's structure.

Two amino acid sequences are “substantially homologous” when at leastabout 70% of the amino acid residues (preferably at least about 80%, andmost preferably at least about 90 or 95%) are identical, or representconservative substitutions.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns, or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to prevent, and preferably reduce by at least about 30percent, preferably by at least 50 percent, preferably by at least 70percent, preferably by at least 80 percent, preferably by at least 90%,a clinically significant change in the growth or progression or mitoticactivity of a target cellular mass, group of cancer cells or tumor, orother feature of pathology. For example, the degree of EGFR activationor activity or amount or number of EGFR positive cells, particularly ofantibody or binding member reactive or positive cells may be reduced.

A DNA sequence is “operatively linked” to an expression control sequencewhen the expression control sequence controls and regulates thetranscription and translation of that DNA sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the DNA sequence to be expressed and maintaining thecorrect reading frame to permit expression of the DNA sequence under thecontrol of the expression control sequence and production of the desiredproduct encoded by the DNA sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted in front of the gene.

The term “standard hybridization conditions” refers to salt andtemperature conditions substantially equivalent to 5×SSC and 65° C. forboth hybridization and wash. However, one skilled in the art willappreciate that such “standard hybridization conditions” are dependenton particular conditions including the concentration of sodium andmagnesium in the buffer, nucleotide sequence length and concentration,percent mismatch, percent formamide, and the like. Also important in thedetermination of “standard hybridization conditions” is whether the twosequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standardhybridization conditions are easily determined by one skilled in the artaccording to well known formulae, wherein hybridization is typically10-20° C. below the predicted or determined T_(m) with washes of higherstringency, if desired.

B. Detailed Disclosure

The present invention provides a novel specific binding member,particularly an antibody or fragment thereof, including immunogenicfragments, which recognizes an EGFR epitope which is found intumorigenic, hyperproliferative or abnormal cells wherein the epitope isenhanced or evident upon aberrant post-translational modification andnot detectable in normal or wild type cells. In a particular butnon-limiting embodiment, the binding member, such as the antibody,recognizes an EGFR epitope which is enhanced or evident upon simplecarbohydrate modification or early glycosylation and is reduced or notevident in the presence of complex carbohydrate modification orglycosylation. The specific binding member, such as the antibody orfragment thereof, does not bind to or recognize normal or wild typecells containing normal or wild type EGFR epitope in the absence ofoverexpression and in the presence of normal EGFR post-translationalmodification.

The invention relates to a specific binding member, particularly anantibody or a fragment thereof, which recognizes an EGFR epitope whichis present in cells expressing amplified EGFR or expressing the de2-7EGFR and not detectable in cells expressing normal or wild type EGFR,particularly in the presence of normal post-translational modification.

It is further noted and herein demonstrated that an additionalnon-limiting observation or characteristic of the antibodies of thepresent invention is their recognition of their epitope in the presenceof high mannose groups, which is a characteristic of early glycosylationor simple carbohydrate modification. Thus, altered or aberrantglycosylation facilitates the presence and/or recognition of theantibody epitope or comprises a portion of the antibody epitope.

Glycosylation includes and encompasses the post-translationalmodification of proteins, termed glycoproteins, by addition ofoligosaccharides. Oligosaccharides are added at glycosylation sites inglycoproteins, particularly including N-linked oligosaccharides andO-linked oligosaccharides. N-linked oligosaccharides are added to an Asnresidue, particularly wherein the Asn residue is in the sequenceN—X—S/T, where X cannot be Pro or Asp, and are the most common onesfound in glycoproteins. In the biosynthesis of N-linked glycoproteins, ahigh mannose type oligosaccharide (generally comprised of dolichol,N-Acetylglucosamine, mannose and glucose is first formed in theendoplasmic reticulum (ER). The high mannose type glycoproteins are thentransported from the ER to the Golgi, where further processing andmodification of the oligosaccharides normally occurs. O-linkedoligosaccharides are added to the hydroxyl group of Ser or Thr residues.In O-linked oligosaccharides, N-Acetylglucosamine is first transferredto the Ser or Thr residue by N-Acetylglucosaminyltransferase in the ER.The protein then moves to the Golgi where further modification and chainelongation occurs.

In a particular aspect of the invention and as stated above, the presentinventors have discovered novel monoclonal antibodies, exemplifiedherein by the antibody designated mAb 806 and its chimeric ch806, whichspecifically recognize amplified wild type EGFR and the de2-7 EGFR, yetbind to an epitope distinct from the unique junctional peptide of thede2-7 EGFR mutation. The antibodies of the present inventionspecifically recognize overexpressed EGFR, including amplified EGFR andmutant EGFR (exemplified herein by the de2-7 mutation), particularlyupon aberrant post-translational modification. Additionally, while mAb806 does not recognize the normal, wild type EGFR expressed on the cellsurface of glioma cells, it does bind to the extracellular domain of theEGFR immobilized on the surface of ELISA plates, indicating aconformational epitope with a polypeptide aspect. Importantly, mAb 806did not bind significantly to normal tissues such as liver and skin,which express levels of endogenous wt EGFR that are higher than in mostother normal tissues, but wherein EGFR is not overexpressed oramplified. Thus, mAb806 demonstrates novel and useful specificity,recognizing de2-7 EGFR and amplified EGFR, while not recognizing normal,wild type EGFR or the unique junctional peptide which is characteristicof de2-7 EGFR.

In a preferred aspect, the antibody is one which has the characteristicsof the antibody which the inventors have identified and characterized,in particular recognizing amplified EGFR and de2-7EGFR. In aparticularly preferred aspect the antibody is the mAb 806, or activefragments thereof. In a further preferred aspect the antibody of thepresent invention comprises the VH and VL amino acid sequences depictedin FIG. 14 (SEQ ID NO:2) and FIG. 15 (SEQ ID NO:4) respectively. Themature amino acid sequences (without the signal sequence) are depicted,respectively, in FIG. 16 with respect to the VH (SEQ ID NO:11) and inFIG. 17 with respect to the VL (SEQ ID NO:12).

Preferably the epitope of the specific binding member or antibody islocated within the region comprising residues 273-501 of the maturenormal or wild type EGFR sequence. Therefore, also provided are specificbinding proteins, such as antibodies, which bind to the de2-7 EGFR at anepitope located within the region comprising residues 273-501 of theEGFR sequence. The epitope may be determined by any conventional epitopemapping techniques known to the person skilled in the art.Alternatively, the DNA sequence encoding residues 273-501 could bedigested, and the resultant fragments expressed in a suitable host.Antibody binding could be determined as mentioned above.

In particular, the member will bind to an epitope comprising residues273-501 of the mature normal or wild type EGFR. However other antibodieswhich show the same or a substantially similar pattern of reactivityalso form an aspect of the invention. This may be determined bycomparing such members with an antibody comprising the VH and VL domainsshown in SEQ ID NO:2 and SEQ ID NO:4 respectively. The comparison willtypically be made using a Western blot in which binding members arebound to duplicate blots prepared from a nuclear preparation of cells sothat the pattern of binding can be directly compared.

In another aspect, the invention provides an antibody capable ofcompeting with the 806 antibody, under conditions in which at least 10%of an antibody having the VH and VL sequences of the 806 antibody isblocked from binding to de2-7EGFR by competition with such an antibodyin an ELISA assay. As set forth above, anti-idiotype antibodies arecontemplated and are illustrated herein.

An isolated polypeptide consisting essentially of the epitope comprisingresidues 273-501 of the mature wild type EGFR (residues 6-234 of maturede2-7 EGFR) forms another aspect of the present invention. The peptideof the invention is particularly useful in diagnostic assays or kits andtherapeutically or prophylactically, including as an anti-tumor oranti-cancer vaccine. Thus compositions of the peptide of the presentinvention include pharmaceutical composition and immunogeniccompositions.

Diagnostic and Therapeutic Uses

The unique specificity of the specific binding members, particularlyantibodies or fragments thereof, of the present invention, whereby thebinding member(s) recognize an EGFR epitope which is found intumorigenic, hyperproliferative or abnormal cells and not detectable innormal or wild type cells and wherein the epitope is enhanced or evidentupon aberrant post-translational modification and wherein the member(s)bind to the de2-7 EGFR and amplified EGFR but not the wt EGFR, providesdiagnostic and therapeutic uses to identify, characterize, target andtreat, reduce or eliminate a number of tumorigenic cell types and tumortypes, for example head and neck, breast, lung, bladder or prostatetumors and glioma, without the problems associated with normal tissueuptake that may be seen with previously known EGFR antibodies. Thus,cells overexpressing EGFR (e.g. by amplification or expression of amutant or variant EGFR), particularly those demonstrating aberrantpost-translational modification may be recognized, isolated,characterized, targeted and treated or eliminated utilizing the bindingmember(s), particularly antibody(ies) or fragments thereof of thepresent invention.

The antibodies of the present invention can thus specifically categorizethe nature of EGFR tumors or tumorigenic cells, by staining or otherwiserecognizing those tumors or cells wherein EGFR overexpression,particularly amplification and/or EGFR mutation, particularly de2-7EGFR,is present. Further, the antibodies of the present invention, asexemplified by mAb 806 and chimeric antibody ch806, demonstratesignificant in vivo anti-tumor activity against tumors containingamplified EGFR and against de2-7 EGFR positive xenografts.

As outlined above, the inventors have found that the specific bindingmember of the invention recognises tumor-associated forms of the EGFR(de2-7 EGFR and amplified EGFR) but not the normal, wild-type receptorwhen expressed in normal cells. It is believed that antibody recognitionis dependent upon an aberrant post-translational modification (e.g., aunique glycosylation, acetylation or phosphorylation variant) of theEGFR expressed in cells exhibiting overexpression of the EGFR gene.

As described below, mAb 806 and ch806 have been used in therapeuticstudies. mAb 806 and ch806 are shown to inhibit growth of overexpressing(e.g. amplified) EGFR xenografts and human de2-7 EGFR expressingxenografts of human tumors and to induce significant necrosis withinsuch tumors.

Moreover, the antibodies of the present invention inhibit the growth ofintracranial tumors in a preventative model. This model involvesinjecting glioma cells expressing de2-7 EGFR into nude mice and theninjecting the antibody intracranially either on the same day or within 1to 3 days, optionally with repeated doses. The doses of antibody aresuitably about 10 μg. Mice injected with antibody are compared tocontrols, and it has been found that survival of the treated mice issignificantly increased.

Therefore, in a further aspect of the invention, there is provided amethod of treatment of a tumor, a cancerous condition, a precancerouscondition, and any condition related to or resulting fromhyperproliferative cell growth comprising administration of a specificbinding member of the invention.

Antibodies of the present invention are designed to be used in methodsof diagnosis and treatment of tumors in human or animal subjects,particularly epithelial tumors. These tumors may be primary or secondarysolid tumors of any type including, but not limited to, glioma, breast,lung, prostate, head or neck tumors.

Binding Member and Antibody Generation

The general methodology for making monoclonal antibodies by hybridomasis well known Immortal, antibody-producing cell lines can also becreated by techniques other than fusion, such as direct transformationof B lymphocytes with oncogenic DNA, or transfection with Epstein-Barrvirus. See, e.g., M. Schreier et al., “Hybridoma Techniques” (1980);Hammerling et al., “Monoclonal Antibodies And T-cell Hybridomas” (1981);Kennett et al., “Monoclonal Antibodies” (1980); see also U.S. Pat. Nos.4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917;4,472,500; 4,491,632; 4,493,890.

Panels of monoclonal antibodies produced against EFGR can be screenedfor various properties; i.e., isotype, epitope, affinity, etc. Ofparticular interest are monoclonal antibodies that mimic the activity ofEFGR or its subunits. Such monoclonals can be readily identified inspecific binding member activity assays. High affinity antibodies arealso useful when immunoaffinity purification of native or recombinantspecific binding member is possible.

Methods for producing polyclonal anti-EFGR antibodies are well-known inthe art. See U.S. Pat. No. 4,493,795 to Nestor et al. A monoclonalantibody, typically containing Fab and/or F(ab′)₂ portions of usefulantibody molecules, can be prepared using the hybridoma technologydescribed in Antibodies—A Laboratory Manual, Harlow and Lane, eds., ColdSpring Harbor Laboratory, New York (1988), which is incorporated hereinby reference. Briefly, to form the hybridoma from which the monoclonalantibody composition is produced, a myeloma or other self-perpetuatingcell line is fused with lymphocytes obtained from the spleen of a mammalhyperimmunized with an appropriate EGFR.

Splenocytes are typically fused with myeloma cells using polyethyleneglycol (PEG) 6000. Fused hybrids are selected by their sensitivity toHAT. Hybridomas producing a monoclonal antibody useful in practicingthis invention are identified by their ability to immunoreact with thepresent antibody or binding member and their ability to inhibitspecified tumorigenic or hyperproliferative activity in target cells.

A monoclonal antibody useful in practicing the present invention can beproduced by initiating a monoclonal hybridoma culture comprising anutrient medium containing a hybridoma that secretes antibody moleculesof the appropriate antigen specificity. The culture is maintained underconditions and for a time period sufficient for the hybridoma to secretethe antibody molecules into the medium. The antibody-containing mediumis then collected. The antibody molecules can then be further isolatedby well-known techniques.

Media useful for the preparation of these compositions are bothwell-known in the art and commercially available and include syntheticculture media, inbred mice and the like. An exemplary synthetic mediumis Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol.8:396 (1959)) supplemented with 4.5 gm/l glucose, 20 mm glutamine, and20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

Methods for producing monoclonal anti-EGFR antibodies are alsowell-known in the art. See Niman et al., Proc. Nall. Acad. Sci. USA,80:4949-4953 (1983). Typically, the EGFR or a peptide analog is usedeither alone or conjugated to an immunogenic carrier, as the immunogenin the before described procedure for producing anti-EGFR monoclonalantibodies. The hybridomas are screened for the ability to produce anantibody that immunoreacts with the EGFR present in tumorigenic,abnormal or hyperproliferative cells. Other anti-EGFR antibodies includebut are not limited to the HuMAX-EGFr antibody from Genmab/Medarex, the108 antibody (ATCC HB9764) and U.S. Pat. No. 6,217,866, and antibody14E1 from Schering A G (U.S. Pat. No. 5,942,602).

Recombinant Binding Members, Chimerics, Bispecifics and Fragments

In general, the CDR3 regions, comprising amino acid sequencessubstantially as set out as the CDR3 regions of SEQ ID NO:2 and SEQ IDNO:4 will be carried in a structure which allows for binding of the CDR3regions to an tumor antigen. In the case of the CDR3 region of SEQ IDNO:4, this is preferably carried by the VL region of SEQ ID NO:4.

By “substantially as set out” it is meant that that CDR3 regions of theinvention will be either identical or highly homologous to the specifiedregions of SEQ ID NO:2 and SEQ ID NO:4. By “highly homologous” it iscontemplated that only a few substitutions, preferably from 1 to 8,preferably from 1 to 5, preferably from 1 to 4, or from 1 to 3 or 1 or 2substitutions may be made in the CDRs.

The structure for carrying the CDR3s of the invention will generally beof an antibody heavy or light chain sequence or substantial portionthereof in which the CDR3 regions are located at locations correspondingto the CDR3 region of naturally occurring VH and VL antibody variabledomains encoded by rearranged immunoglobulin genes. The structures andlocations of immunoglobulin variable domains may be determined byreference to Kabat, E. A. et al, Sequences of Proteins of ImmunologicalInterest. 4th Edition. US Department of Health and Human Services. 1987,and updates thereof.

Preferably, the amino acid sequence substantially as set out as residues93-102 of SEQ ID NO:2 is carried as the CDR3 in a human heavy chainvariable domain or a substantial portion thereof, and the amino acidsequences substantially as set out as residues 24-34, 50-56 and 89-97 ofSEQ ID NO:4 are carried as the CDRs 1-3 respectively in a human lightchain variable domain or a substantial portion thereof.

The variable domains may be derived from any germline or rearrangedhuman variable domain, or may be a synthetic variable domain based onconsensus sequences of known human variable domains. The CDR3-derivedsequences of the invention, as defined in the preceding paragraph, maybe introduced into a repertoire of variable domains lacking CDR3regions, using recombinant DNA technology.

For example, Marks et al (Bio/Technology, 1992, 10:779-783) describemethods of producing repertoires of antibody variable domains in whichconsensus primers directed at or adjacent to the 5′ end of the variabledomain area are used in conjunction with consensus primers to the thirdframework region of human VH genes to provide a repertoire of VHvariable domains lacking a CDR3. Marks et al further describe how thisrepertoire may be combined with a CDR3 of a particular antibody. Usinganalogous techniques, the CDR3-derived sequences of the presentinvention may be shuffled with repertoires of VH or VL domains lacking aCDR3, and the shuffled complete VH or VL domains combined with a cognateVL or VH domain to provide specific binding members of the invention.The repertoire may then be displayed in a suitable host system such asthe phage display system of WO92/01047 so that suitable specific bindingmembers may be selected. A repertoire may consist of from anything from10⁴ individual members upwards, for example from 10⁶ to 10⁸ or 10¹⁰members.

Analogous shuffling or combinatorial techniques are also disclosed byStemmer (Nature, 1994, 370:389-391), who describes the technique inrelation to a β-lactamase gene but observes that the approach may beused for the generation of antibodies.

A further alternative is to generate novel VH or VL regions carrying theCDR3-derived sequences of the invention using random mutagenesis of, forexample, the mAb806 VH or VL genes to generate mutations within theentire variable domain. Such a technique is described by Gram et al(1992, Proc. Natl. Acad. Sci., USA, 89:3576-3580), who used error-pronePCR.

Another method which may be used is to direct mutagenesis to CDR regionsof VH or VL genes. Such techniques are disclosed by Barbas et al, (1994,Proc. Natl. Acad. Sci., USA, 91:3809-3813) and Schier et al (1996, J.Mol. Biol. 263:551-567).

All the above described techniques are known as such in the art and inthemselves do not form part of the present invention. The skilled personwill be able to use such techniques to provide specific binding membersof the invention using routine methodology in the art.

A substantial portion of an immunoglobulin variable domain will compriseat least the three CDR regions, together with their interveningframework regions. Preferably, the portion will also include at leastabout 50% of either or both of the first and fourth framework regions,the 50% being the C-terminal 50% of the first framework region and theN-terminal 50% of the fourth framework region. Additional residues atthe N-terminal or C-terminal end of the substantial part of the variabledomain may be those not normally associated with naturally occurringvariable domain regions. For example, construction of specific bindingmembers of the present invention made by recombinant DNA techniques mayresult in the introduction of N- or C-terminal residues encoded bylinkers introduced to facilitate cloning or other manipulation steps.Other manipulation steps include the introduction of linkers to joinvariable domains of the invention to further protein sequences includingimmunoglobulin heavy chains, other variable domains (for example in theproduction of diabodies) or protein labels as discussed in more detailbelow.

Although in a preferred aspect of the invention specific binding memberscomprising a pair of binding domains based on sequences substantiallyset out in SEQ ID NO:2 and SEQ ID NO:4 are preferred, single bindingdomains based on either of these sequences form further aspects of theinvention. In the case of the binding domains based on the sequencesubstantially set out in SEQ ID NO:2, such binding domains may be usedas targeting agents for tumor antigens since it is known thatimmunoglobulin VH domains are capable of binding target antigens in aspecific manner.

In the case of either of the single chain specific binding domains,these domains may be used to screen for complementary domains capable offorming a two-domain specific binding member which has in vivoproperties as good as or equal to the mAb806 antibody disclosed herein.

This may be achieved by phage display screening methods using theso-called hierarchical dual combinatorial approach as disclosed in U.S.Pat. No. 5,969,108 in which an individual colony containing either an Hor L chain clone is used to infect a complete library of clones encodingthe other chain (L or H) and the resulting two-chain specific bindingmember is selected in accordance with phage display techniques such asthose described in that reference. This technique is also disclosed inMarks et al, ibid.

Specific binding members of the present invention may further compriseantibody constant regions or parts thereof. For example, specificbinding members based on SEQ ID NO:4 may be attached at their C-terminalend to antibody light chain constant domains including human Cκ or Cλchains, preferably Cλ chains. Similarly, specific binding members basedon SEQ ID NO:2 may be attached at their C-terminal end to all or part ofan immunoglobulin heavy chain derived from any antibody isotype, e.g.IgG, IgA, IgE, IgD and IgM and any of the isotype sub-classes,particularly IgG1, IgG2b, and IgG4. IgG1 is preferred.

The advent of monoclonal antibody (mAb) technology 25 years ago hasprovide an enormous repertoire of useful research reagents and createdthe opportunity to use antibodies as approved pharmaceutical reagents incancer therapy, autoimmune disorders, transplant rejection, antiviralprophylaxis and as anti-thrombotics (Glennie and Johnson 2000). Theapplication of molecular engineering to convert murine mAbs intochimeric mAbs (mouse V-region, human C-region) and humanised reagentswhere only the mAb complementarity-determining regions (CDR) are ofmurine origin has been critical to the clinical success of mAb therapy.The engineered mAbs have markedly reduced or absent immunogenicity,increased serum half-life and the human Fc portion of the mAb increasesthe potential to recruit the immune effectors of complement andcytotoxic cells (Clark 2000). Investigations into the biodistribution,pharmacokinetics and any induction of an immune response to clinicallyadministered mAbs requires the development of analyses to discriminatebetween the pharmaceutical and endogenous proteins.

The antibodies, or any fragments thereof, may also be conjugated orrecombinantly fused to any cellular toxin, bacterial or other, e.g.pseudomonas exotoxin, ricin, or diphtheria toxin. The part of the toxinused can be the whole toxin, or any particular domain of the toxin. Suchantibody-toxin molecules have successfully been used for targeting andtherapy of different kinds of cancers, see e.g. Pastan, Biochim BiophysActa. 1997 Oct. 24; 1333(2):C1-6; Kreitman et al., N Engl J. Med. 2001Jul. 26; 345(4):241-7; Schnell et al., Leukemia. 2000 January;14(1):129-35; Ghetie et al., Mol. Biotechnol. 2001 July; 18(3):251-68.

Bi- and tri-specific multimers can be formed by association of differentscFv molecules and have been designed as cross-linking reagents forT-cell recruitment into tumors (immunotherapy), viral retargeting (genetherapy) and as red blood cell agglutination reagents(immunodiagnostics), see e.g. Todorovska et al., J Immunol Methods. 2001Feb. 1; 248(1-2):47-66; Tomlinson et al., Methods Enzymol. 2000;326:461-79; McCall et al., J. Immunol. 2001 May 15; 166(10):6112-7.

Fully human antibodies can be prepared by immunizing transgenic micecarrying large portions of the human immunoglobulin heavy and lightchains. These mice, examples of such mice are the Xenomouse™ (Abgenix,Inc.) (U.S. Pat. Nos. 6,075,181 and 6,150,584), the HuMAb-Mouse™(Medarex, Inc./GenPharm) (U.S. Pat. Nos. 5,545,806 and 5,569,825), theTransChromo Mouse™ (Kirin) and the KM Mouse™ (Medarex/Kirin), are wellknown within the art. Antibodies can then be prepared by, e.g. standardhybridoma technique or by phage display. These antibodies will thencontain only fully human amino acid sequences.

Fully human antibodies can also be generated using phage display fromhuman libraries. Phage display may be performed using methods well knownto the skilled artisan, as in Hoogenboom et al and Marks et al(Hoogenboom H R and Winter G. (1992) J Mol. Biol. 227(2):381-8; Marks JD et al (1991) J Mol. Biol. 222(3):581-97; and also U.S. Pat. Nos.5,885,793 and 5,969,108).

Therapeutic Antibodies and Uses

The in vivo properties, particularly with regard to tumor:blood ratioand rate of clearance, of specific binding members of the invention willbe at least comparable to mAb806. Following administration to a human oranimal subject such a specific binding member will show a peak tumor toblood ratio of >1:1. Preferably at such a ratio the specific bindingmember will also have a tumor to organ ratio of greater than 1:1,preferably greater than 2:1, more preferably greater than 5:1.Preferably at such a ratio the specific binding member will also have anorgan to blood ratio of <1:1 in organs away from the site of the tumor.These ratios exclude organs of catabolism and secretion of theadministered specific binding member. Thus in the case of scFvs and Fabs(as shown in the accompanying examples), the binding members aresecreted via the kidneys and there is greater presence here than otherorgans. In the case of whole IgGs, clearance will be at least in part,via the liver. The peak localisation ratio of the intact antibody willnormally be achieved between 10 and 200 hours following administrationof the specific binding member. More particularly, the ratio may bemeasured in a tumor xenograft of about 0.2-1.0 g formed subcutaneouslyin one flank of an athymic nude mouse.

Antibodies of the invention may be labelled with a detectable orfunctional label. Detectable labels include, but are not limited to,radiolabels such as the isotopes ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co,⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²¹I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹¹¹In, ²¹¹At, ¹⁹⁸Au, ⁶⁷Cu,²²⁵Ac, ²¹³Bi, ⁹⁹Tc and ¹⁸⁶Re, which may be attached to antibodies of theinvention using conventional chemistry known in the art of antibodyimaging. Labels also include fluorescent labels and labels usedconventionally in the art for MRI-CT imagine. They also include enzymelabels such as horseradish peroxidase. Labels further include chemicalmoieties such as biotin which may be detected via binding to a specificcognate detectable moiety, e.g. labelled avidin.

Functional labels include substances which are designed to be targetedto the site of a tumor to cause destruction of tumor tissue. Suchfunctional labels include cytotoxic drugs such as 5-fluorouracil orricin and enzymes such as bacterial carboxypeptidase or nitroreductase,which are capable of converting prodrugs into active drugs at the siteof a tumor.

Also, antibodies including both polyclonal and monoclonal antibodies,and drugs that modulate the production or activity of the specificbinding members, antibodies and/or their subunits may possess certaindiagnostic applications and may for example, be utilized for the purposeof detecting and/or measuring conditions such as cancer, precancerouslesions, conditions related to or resulting from hyperproliferative cellgrowth or the like. For example, the specific binding members,antibodies or their subunits may be used to produce both polyclonal andmonoclonal antibodies to themselves in a variety of cellular media, byknown techniques such as the hybridoma technique utilizing, for example,fused mouse spleen lymphocytes and myeloma cells. Likewise, smallmolecules that mimic or antagonize the activity(ies) of the specificbinding members of the invention may be discovered or synthesized, andmay be used in diagnostic and/or therapeutic protocols.

The radiolabelled specific binding members, particularly antibodies andfragments thereof, are useful in in vitro diagnostics techniques and inin vivo radioimaging techniques and in radioimmunotherapy. In theinstance of in vivo imaging, the specific binding members of the presentinvention may be conjugated to an imaging agent rather than aradioisotope(s), including but not limited to a magnetic resonance imageenhancing agent, wherein for instance an antibody molecule is loadedwith a large number of paramagnetic ions through chelating groups.Examples of chelating groups include EDTA, porphyrins, polyamines crownethers and polyoximes. Examples of paramagnetic ions include gadolinium,iron, manganese, rhenium, europium, lanthanum, holmium and ferbium. In afurther aspect of the invention, radiolabelled specific binding members,particularly antibodies and fragments thereof, particularlyradioimmunoconjugates, are useful in radioimmunotherapy, particularly asradiolabelled antibodies for cancer therapy. In a still further aspect,the radiolabelled specific binding members, particularly antibodies andfragments thereof, are useful in radioimmuno-guided surgery techniques,wherein they can identify and indicate the presence and/or location ofcancer cells, precancerous cells, tumor cells, and hyperproliferativecells, prior to, during or following surgery to remove such cells.

Immunoconjugates or antibody fusion proteins of the present invention,wherein the specific binding members, particularly antibodies andfragments thereof, of the present invention are conjugated or attachedto other molecules or agents further include, but are not limited tobinding members conjugated to a chemical ablation agent, toxin,immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent ordrug.

Radioimmunotherapy (RAIT) has entered the clinic and demonstratedefficacy using various antibody immunoconjugates. ¹³¹I labeled humanizedanti-carcinoembryonic antigen (anti-CEA) antibody hMN-14 has beenevaluated in colorectal cancer (Behr T M et al (2002) Cancer94(4Suppl):1373-81) and the same antibody with ⁹⁰Y label has beenassessed in medullary thyroid carcinoma (Stein R et al (2002) Cancer94(1):51-61). Radioimmunotherapy using monoclonal antibodies has alsobeen assessed and reported for non-Hodgkin's lymphoma and pancreaticcancer (Goldenberg D M (2001) Crit Rev Oncol Hematol 39(1-2):195-201;Gold D V et al (2001) Crit Rev Oncol Hematol 39 (1-2) 147-54).Radioimmunotherapy methods with particular antibodies are also describedin U.S. Pat. Nos. 6,306,393 and 6,331,175. Radioimmunoguided surgery(RIGS) has also entered the clinic and demonstrated efficacy andusefulness, including using anti-CEA antibodies and antibodies directedagainst tumor-associated antigens (Kim J C et al (2002) Int J Cancer97(4):542-7; Schneebaum S et al (2001) World J Surg 25(12):1495-8;Avital S et al (2000) Cancer 89(8):1692-8; McIntosh D G et al (1997)Cancer Biother Radiopharm 12 (4):287-94).

Antibodies of the present invention may be administered to a patient inneed of treatment via any suitable route, usually by injection into thebloodstream or CSF, or directly into the site of the tumor. The precisedose will depend upon a number of factors, including whether theantibody is for diagnosis or for treatment, the size and location of thetumor, the precise nature of the antibody (whether whole antibody,fragment, diabody, etc), and the nature of the detectable or functionallabel attached to the antibody. Where a radionuclide is used fortherapy, a suitable maximum single dose is about 45 mCi/m², to a maximumof about 250 mCi/m². Preferable dosage is in the range of 15 to 40 mCi,with a further preferred dosage range of 20 to 30 mCi, or 10 to 30 mCi.Such therapy may require bone marrow or stem cell replacement. A typicalantibody dose for either tumor imaging or tumor treatment will be in therange of from 0.5 to 40 mg, preferably from 1 to 4 mg of antibody inF(ab′)2 form. Naked antibodies are preferable administered in doses of20 to 1000 mg protein per dose, or 20 to 500 mg protein per dose, or 20to 100 mg protein per dose. This is a dose for a single treatment of anadult patient, which may be proportionally adjusted for children andinfants, and also adjusted for other antibody formats in proportion tomolecular weight. Treatments may be repeated at daily, twice-weekly,weekly or monthly intervals, at the discretion of the physician.

These formulations may include a second binding protein, such as theEGFR binding proteins described supra. In an especially preferred form,this second binding protein is a monoclonal antibody such as 528 or 225,discussed infra.

Pharmaceutical and Therapeutic Compositions

Specific binding members of the present invention will usually beadministered in the form of a pharmaceutical composition, which maycomprise at least one component in addition to the specific bindingmember.

Thus pharmaceutical compositions according to the present invention, andfor use in accordance with the present invention, may comprise, inaddition to active ingredient, a pharmaceutically acceptable excipient,carrier, buffer, stabiliser or other materials well known to thoseskilled in the art. Such materials should be non-toxic and should notinterfere with the efficacy of the active ingredient. The precise natureof the carrier or other material will depend on the route ofadministration, which may be oral, or by injection, e.g. intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally comprise a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil.

Physiological saline solution, dextrose or other saccharide solution orglycols such as ethylene glycol, propylene glycol or polyethylene glycolmay be included.

For intravenous, injection, or injection at the site of affliction, theactive ingredient will be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pH, isotonicityand stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilisers, buffers, antioxidants and/orother additives may be included, as required.

A composition may be administered alone or in combination with othertreatments, therapeutics or agents, either simultaneously orsequentially dependent upon the condition to be treated. In addition,the present invention contemplates and includes compositions comprisingthe binding member, particularly antibody or fragment thereof, hereindescribed and other agents or therapeutics such as anti-cancer agents ortherapeutics, hormones, anti-EGFR agents or antibodies, or immunemodulators. More generally these anti-cancer agents may be tyrosinekinase inhibitors or phosphorylation cascade inhibitors,post-translational modulators, cell growth or division inhibitors (e.g.anti-mitotics), or signal transduction inhibitors. Other treatments ortherapeutics may include the administration of suitable doses of painrelief drugs such as non-steroidal anti-inflammatory drugs (e.g.aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such asmorphine, or anti-emetics. The composition can be administered incombination (either sequentially (i.e. before or after) orsimultaneously) with tyrosine kinase inhibitors (including, but notlimited to AG1478 and ZD1839, STI571, OSI-774, SU-6668), doxorubicin,temozolomide, cisplatin, carboplatin, nitrosoureas, procarbazine,vincristine, hydroxyurea, 5-fluoruracil, cytosine arabinoside,cyclophosphamide, epipodophyllotoxin, carmustine, lomustine, and/orother chemotherapeutic agents. Thus, these agents may be anti-EGFRspecific agents, or tyrosine kinase inhibitors such as AG1478, ZD1839,STI571, OSI-774, or SU-6668 or may be more general anti-cancer andanti-neoplastic agents such as doxorubicin, cisplatin, temozolomide,nitrosoureas, procarbazine, vincristine, hydroxyurea, 5-fluoruracil,cytosine arabinoside, cyclophosphamide, epipodophyllotoxin, carmustine,or lomustine. In addition, the composition may be administered withhormones such as dexamethasone, immune modulators, such as interleukins,tumor necrosis factor (TNF) or other growth factors or cytokines whichstimulate the immune response and reduction or elimination of cancercells or tumors. An immune modulator such as TNF may be combinedtogether with a member of the invention in the form of a bispecificantibody recognizing the 806 EGFR epitope as well as binding to TNFreceptors. The composition may also be administered with, or may includecombinations along with other anti-EGFR antibodies, including but notlimited to the anti-EGFR antibodies 528, 225, SC-03, DH8.3, L8A4, Y10,ICR62 and ABX-EGF.

Previously the use of agents such as doxorubicin and cisplatin inconjunction with anti-EGFR antibodies have produced enhanced anti-tumoractivity (Fan et al, 1993; Baselga et al, 1993). The combination ofdoxorubicin and mAb 528 resulted in total eradication of establishedA431 xenografts, whereas treatment with either agent alone caused onlytemporary in vivo growth inhibition (Baselga et al, 1993). Likewise, thecombination of cisplatin and either mAb 528 or 225 also led to theeradication of well established A431 xenografts, which was not observedwhen treatment with either agent was used (Fan et al, 1993).

Conventional Radiotherapy

In addition, the present invention contemplates and includes therapeuticcompositions for the use of the binding member in combination withconventional radiotherapy. It has been indicated that treatment withantibodies targeting EGF receptors can enhance the effects ofconventional radiotherapy (Milas et al., Clin Cancer Res. 2000February:6 (2):701 8, Huang et al., Clin Cancer Res. 2000 June:6(6):2166 74).

As demonstrated herein, combinations of the binding member of thepresent invention, particularly an antibody or fragment thereof,preferably the mAb806, ch806 or a fragment thereof, and anti-cancertherapeutics, particularly anti-EGFR therapeutics, including otheranti-EGFR antibodies, demonstrate effective therapy, and particularlysynergy, against xenografted tumors. In the examples, it is demonstratedthat the combination of AG1478 and mAb 806 results in significantlyenhanced reduction of A431 xenograft tumor volume in comparison withtreatment with either agent alone. AG1478(4-(3-chloroanilino)-6,7-dimethoxyquinazoline) is a potent and selectiveinhibitor of the EGF receptor kinase and is particularly described inU.S. Pat. No. 5,457,105, incorporated by reference herein in itsentirety (see also, Liu, W. et al (1999) J. Cell Sci. 112:2409; Eguchi,S. et al (1998) J. Biol. Chem. 273:8890; Levitsky, A. and Gazit, A.(1995) Science 267:1782). The specification examples further demonstratetherapeutic synergy of the 806 antibody with other anti-EGFR antibodies,particularly with the 528 anti-EGFR antibody.

The present invention further contemplates therapeutic compositionsuseful in practicing the therapeutic methods of this invention. Asubject therapeutic composition includes, in admixture, apharmaceutically acceptable excipient (carrier) and one or more of aspecific binding member, polypeptide analog thereof or fragment thereof,as described herein as an active ingredient. In a preferred embodiment,the composition comprises an antigen capable of modulating the specificbinding of the present binding member/antibody with a target cell.

The preparation of therapeutic compositions which contain polypeptides,analogs or active fragments as active ingredients is well understood inthe art. Typically, such compositions are prepared as injectables,either as liquid solutions or suspensions. However, solid forms suitablefor solution in, or suspension in, liquid prior to injection can also beprepared. The preparation can also be emulsified. The active therapeuticingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanol,or the like and combinations thereof. In addition, if desired, thecomposition can contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents which enhance theeffectiveness of the active ingredient.

A polypeptide, analog or active fragment can be formulated into thetherapeutic composition as neutralized pharmaceutically acceptable saltforms. Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide or antibodymolecule) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed from thefree carboxyl groups can also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

The therapeutic polypeptide-, analog- or active fragment-containingcompositions are conventionally administered intravenously, as byinjection of a unit dose, for example. The term “unit dose” when used inreference to a therapeutic composition of the present invention refersto physically discrete units suitable as unitary dosage for humans, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired diluent; i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's immune system to utilize the active ingredient, and degree ofEFGR binding capacity desired. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitionerand are peculiar to each individual. However, suitable dosages may rangefrom about 0.1 to 20, preferably about 0.5 to about 10, and morepreferably one to several, milligrams of active ingredient per kilogrambody weight of individual per day and depend on the route ofadministration. Suitable regimes for initial administration and boostershots are also variable, but are typified by an initial administrationfollowed by repeated doses at one or more hour intervals by a subsequentinjection or other administration. Alternatively, continuous intravenousinfusion sufficient to maintain concentrations of ten nanomolar to tenmicromolar in the blood are contemplated.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally comprise a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, injection, or injection at the site of affliction, theactive ingredient will be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pH, isotonicityand stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilisers, buffers, antioxidants and/orother additives may be included, as required.

Diagnostic Assays

The present invention also relates to a variety of diagnosticapplications, including methods for detecting the presence of stimulisuch as aberrantly expressed EGFR, by reference to their ability to berecognized by the present specific binding member. As mentioned earlier,the EGFR can be used to produce antibodies to itself by a variety ofknown techniques, and such antibodies could then be isolated andutilized as in tests for the presence of particular EGFR activity insuspect target cells.

Diagnostic applications of the specific binding members of the presentinvention, particularly antibodies and fragments thereof, include invitro and in vivo applications well known and standard to the skilledartisan and based on the present description. Diagnostic assays and kitsfor in vitro assessment and evaluation of EGFR status, particularly withregard to aberrant expression of EGFR, may be utilized to diagnose,evaluate and monitor patient samples including those known to have orsuspected of having cancer, a precancerous condition, a conditionrelated to hyperproliferative cell growth or from a tumor sample. Theassessment and evaluation of EGFR status is also useful in determiningthe suitability of a patient for a clinical trial of a drug or for theadministration of a particular chemotherapeutic agent or specificbinding member, particularly an antibody, of the present invention,including combinations thereof, versus a different agent or bindingmember. This type of diagnostic monitoring and assessment is already inpractice utilizing antibodies against the HER2 protein in breast cancer(Hercep Test, Dako Corporation), where the assay is also used toevaluate patients for antibody therapy using Herceptin. In vivoapplications include imaging of tumors or assessing cancer status ofindividuals, including radioimaging.

As suggested earlier, the diagnostic method of the present inventioncomprises examining a cellular sample or medium by means of an assayincluding an effective amount of an antagonist to an EFGR/protein, suchas an anti-EFGR antibody, preferably an affinity-purified polyclonalantibody, and more preferably a mAb. In addition, it is preferable forthe anti-EFGR antibody molecules used herein be in the form of Fab,Fab′, F(ab′)₂ or F(v) portions or whole antibody molecules. Aspreviously discussed, patients capable of benefiting from this methodinclude those suffering from cancer, a pre-cancerous lesion, a viralinfection, pathologies involving or resulting from hyperproliferativecell growth or other like pathological derangement. Methods forisolating EFGR and inducing anti-EFGR antibodies and for determining andoptimizing the ability of anti-EFGR antibodies to assist in theexamination of the target cells are all well-known in the art.

Preferably, the anti-EFGR antibody used in the diagnostic methods ofthis invention is an affinity purified polyclonal antibody. Morepreferably, the antibody is a monoclonal antibody (mAb). In addition,the anti-EFGR antibody molecules used herein can be in the form of Fab,Fab′, F(ab′)₂ or F(v) portions of whole antibody molecules.

As described in detail above, antibody(ies) to the EGFR can be producedand isolated by standard methods including the well known hybridomatechniques. For convenience, the antibody(ies) to the EGFR will bereferred to herein as Ab₁ and antibody(ies) raised in another species asAb₂.

The presence of EGFR in cells can be ascertained by the usual in vitroor in vivo immunological procedures applicable to such determinations. Anumber of useful procedures are known. Three such procedures which areespecially useful utilize either the EGFR labeled with a detectablelabel, antibody Ab₁ labeled with a detectable label, or antibody Ab₂labeled with a detectable label. The procedures may be summarized by thefollowing equations wherein the asterisk indicates that the particle islabeled, and “R” stands for the EGFR:R*+Ab ₁ =R*Ab ₁  A.R+Ab*=RAb ₁*  B.R+Ab ₁ +Ab ₂ *=RAb ₁ Ab ₂*  C.

The procedures and their application are all familiar to those skilledin the art and accordingly may be utilized within the scope of thepresent invention. The “competitive” procedure, Procedure A, isdescribed in U.S. Pat. Nos. 3,654,090 and 3,850,752. Procedure C, the“sandwich” procedure, is described in U.S. Pat. Nos. RE 31,006 and4,016,043. Still other procedures are known such as the “doubleantibody,” or “DASP” procedure.

In each instance above, the EGFR forms complexes with one or moreantibody(ies) or binding partners and one member of the complex islabeled with a detectable label.

The fact that a complex has formed and, if desired, the amount thereof,can be determined by known methods applicable to the detection oflabels.

It will be seen from the above, that a characteristic property of Ab₂ isthat it will react with Ab₁. This is because Ab₁ raised in one mammalianspecies has been used in another species as an antigen to raise theantibody Ab₂. For example, Ab₂ may be raised in goats using rabbitantibodies as antigens. Ab₂ therefore would be anti-rabbit antibodyraised in goats. For purposes of this description and claims, Ab₁ willbe referred to as a primary or anti-EGFR antibody, and Ab₂ will bereferred to as a secondary or anti-Ab₁ antibody.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others.

A number of fluorescent materials are known and can be utilized aslabels. These include, for example, fluorescein, rhodamine, auramine,Texas Red®, AMCA™ blue and Lucifer Yellow. A particular detectingmaterial is anti-rabbit antibody prepared in goats and conjugated withfluorescein through an isothiocyanate.

The EGFR or its binding partner(s) such as the present specific bindingmember, can also be labeled with a radioactive element or with anenzyme. The radioactive label can be detected by any of the currentlyavailable counting procedures. The preferred isotope may be selectedfrom ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²¹I, ¹²⁴I,¹²⁵I, ¹³¹I, ¹¹¹In, ²¹¹At, ¹⁹⁸Au, ⁶⁷Cu, ²²⁵Ac, ²¹³Bi, ⁹⁹Tc and ¹⁸⁶Re.

Enzyme labels are likewise useful, and can be detected by any of thepresently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques. Theenzyme is conjugated to the selected particle by reaction with bridgingmolecules such as carbodiimides, diisocyanates, glutaraldehyde and thelike. Many enzymes which can be used in these procedures are known andcan be utilized. The preferred are peroxidase, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090;3,850,752; and 4,016,043 are referred to by way of example for theirdisclosure of alternate labeling material and methods.

A particular assay system that may be advantageously utilized inaccordance with the present invention, is known as a receptor assay. Ina receptor assay, the material to be assayed such as the specificbinding member, is appropriately labeled and then certain cellular testcolonies are inoculated with a quantity of both the labeled andunlabeled material after which binding studies are conducted todetermine the extent to which the labeled material binds to the cellreceptors. In this way, differences in affinity between materials can beascertained.

Accordingly, a purified quantity of the specific binding member may beradiolabeled and combined, for example, with antibodies or otherinhibitors thereto, after which binding studies would be carried out.Solutions would then be prepared that contain various quantities oflabeled and unlabeled uncombined specific binding member, and cellsamples would then be inoculated and thereafter incubated. The resultingcell monolayers are then washed, solubilized and then counted in a gammacounter for a length of time sufficient to yield a standard error of<5%. These data are then subjected to Scatchard analysis after whichobservations and conclusions regarding material activity can be drawn.While the foregoing is exemplary, it illustrates the manner in which areceptor assay may be performed and utilized, in the instance where thecellular binding ability of the assayed material may serve as adistinguishing characteristic.

An assay useful and contemplated in accordance with the presentinvention is known as a “cis/trans” assay. Briefly, this assay employstwo genetic constructs, one of which is typically a plasmid thatcontinually expresses a particular receptor of interest when transfectedinto an appropriate cell line, and the second of which is a plasmid thatexpresses a reporter such as luciferase, under the control of areceptor/ligand complex. Thus, for example, if it is desired to evaluatea compound as a ligand for a particular receptor, one of the plasmidswould be a construct that results in expression of the receptor in thechosen cell line, while the second plasmid would possess a promoterlinked to the luciferase gene in which the response element to theparticular receptor is inserted. If the compound under test is anagonist for the receptor, the ligand will complex with the receptor, andthe resulting complex will bind the response element and initiatetranscription of the luciferase gene. The resulting chemiluminescence isthen measured photometrically, and dose response curves are obtained andcompared to those of known ligands. The foregoing protocol is describedin detail in U.S. Pat. No. 4,981,784 and PCT International PublicationNo. WO 88/03168, for which purpose the artisan is referred.

In a further embodiment of this invention, commercial test kits suitablefor use by a medical specialist may be prepared to determine thepresence or absence of aberrant expression of EGFR, including but notlimited to amplified EGFR and/or an EGFR mutation, in suspected targetcells. In accordance with the testing techniques discussed above, oneclass of such kits will contain at least the labeled EGFR or its bindingpartner, for instance an antibody specific thereto, and directions, ofcourse, depending upon the method selected, e.g., “competitive,”“sandwich,” “DASP” and the like. The kits may also contain peripheralreagents such as buffers, stabilizers, etc.

Accordingly, a test kit may be prepared for the demonstration of thepresence or capability of cells for aberrant expression orpost-translational modification of EGFR, comprising:

-   -   (a) a predetermined amount of at least one labeled        immunochemically reactive component obtained by the direct or        indirect attachment of the present specific binding member or a        specific binding partner thereto, to a detectable label;    -   (b) other reagents; and    -   (c) directions for use of said kit.

More specifically, the diagnostic test kit may comprise:

-   -   (a) a known amount of the specific binding member as described        above (or a binding partner) generally bound to a solid phase to        form an immunosorbent, or in the alternative, bound to a        suitable tag, or plural such end products, etc. (or their        binding partners) one of each;    -   (b) if necessary, other reagents; and    -   (c) directions for use of said test kit.

In a further variation, the test kit may be prepared and used for thepurposes stated above, which operates according to a predeterminedprotocol (e.g. “competitive,” “sandwich,” “double antibody,” etc.), andcomprises:

-   -   (a) a labeled component which has been obtained by coupling the        specific binding member to a detectable label;    -   (b) one or more additional immunochemical reagents of which at        least one reagent is a ligand or an immobilized ligand, which        ligand is selected from the group consisting of:        -   (i) a ligand capable of binding with the labeled component            (a);        -   (ii) a ligand capable of binding with a binding partner of            the labeled component (a);        -   (iii) a ligand capable of binding with at least one of the            component(s) to be determined; and        -   (iv) a ligand capable of binding with at least one of the            binding partners of at least one of the component(s) to be            determined; and    -   (c) directions for the performance of a protocol for the        detection and/or determination of one or more components of an        immunochemical reaction between the EFGR, the specific binding        member, and a specific binding partner thereto.

In accordance with the above, an assay system for screening potentialdrugs effective to modulate the activity of the EFGR, the aberrantexpression or post-translational modification of the EGFR, and/or theactivity or binding of the specific binding member may be prepared. Thereceptor or the binding member may be introduced into a test system, andthe prospective drug may also be introduced into the resulting cellculture, and the culture thereafter examined to observe any changes inthe S-phase activity of the cells, due either to the addition of theprospective drug alone, or due to the effect of added quantities of theknown agent(s).

Nucleic Acids

The present invention further provides an isolated nucleic acid encodinga specific binding member of the present invention. Nucleic acidincludes DNA and RNA. In a preferred aspect, the present inventionprovides a nucleic acid which codes for a polypeptide of the inventionas defined above, including a polypeptide as set out as residues 93-102of SEQ ID NO:2 or 26-35A, 49-64 and 93-102 of SEQ ID NO:2, a polypeptideas set out in residues 24-34, 50-56 and 89-97 of SEQ ID NO:4, and theentire polypeptides of SEQ ID NO:2 and SEQ ID NO:4.

The present invention also provides constructs in the form of plasmids,vectors, transcription or expression cassettes which comprise at leastone polynucleotide as above.

The present invention also provides a recombinant host cell whichcomprises one or more constructs as above. A nucleic acid encoding anyspecific binding member as provided itself forms an aspect of thepresent invention, as does a method of production of the specificbinding member which method comprises expression from encoding nucleicacid therefor. Expression may conveniently be achieved by culturingunder appropriate conditions recombinant host cells containing thenucleic acid. Following production by expression a specific bindingmember may be isolated and/or purified using any suitable technique,then used as appropriate.

Specific binding members and encoding nucleic acid molecules and vectorsaccording to the present invention may be provided isolated and/orpurified, e.g. from their natural environment, in substantially pure orhomogeneous form, or, in the case of nucleic acid, free or substantiallyfree of nucleic acid or genes origin other than the sequence encoding apolypeptide with the required function. Nucleic acid according to thepresent invention may comprise DNA or RNA and may be wholly or partiallysynthetic.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary cells, HeLa cells, baby hamster kidneycells, NSO mouse melanoma cells and many others. A common, preferredbacterial host is E. coli.

The expression of antibodies and antibody fragments in prokaryotic cellssuch as E. coli is well established in the art. For a review, see forexample Pliickthun, A. Bio/Technology 9: 545-551 (1991). Expression ineukaryotic cells in culture is also available to those skilled in theart as an option for production of a specific binding member, see forrecent reviews, for example Raff, M.E. (1993) Curr. Opinion Biotech. 4:573-576; Trill J. J. et al. (1995) Curr. Opinion Biotech 6: 553-560.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids, viral e.g.‘phage, or phagemid, as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrooket al., 1989, Cold Spring Harbor Laboratory Press. Many known techniquesand protocols for manipulation of nucleic acid, for example inpreparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Short Protocols in MolecularBiology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992.The disclosures of Sambrook et al. and Ausubel et al. are incorporatedherein by reference.

Thus, a further aspect of the present invention provides a host cellcontaining nucleic acid as disclosed herein. A still further aspectprovides a method comprising introducing such nucleic acid into a hostcell. The introduction may employ any available technique. Foreukaryotic cells, suitable techniques may include calcium phosphatetransfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia or, for insect cells, baculovirus. For bacterial cells,suitable techniques may include calcium chloride transformation,electroporation and transfection using bacteriophage.

The introduction may be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells under conditions forexpression of the gene.

In one embodiment, the nucleic acid of the invention is integrated intothe genome (e.g. chromosome) of the host cell. Integration may bepromoted by inclusion of sequences which promote recombination with thegenome, in accordance with standard techniques.

The present invention also provides a method which comprises using aconstruct as stated above in an expression system in order to express aspecific binding member or polypeptide as above.

As stated above, the present invention also relates to a recombinant DNAmolecule or cloned gene, or a degenerate variant thereof, which encodesa specific binding member, particularly antibody or a fragment thereof,that possesses an amino acid sequence set forth in SEQ ID NO:2 and/orSEQ ID NO:4; preferably a nucleic acid molecule, in particular arecombinant DNA molecule or cloned gene, encoding the binding member orantibody has a nucleotide sequence or is complementary to a DNA sequenceprovided in SEQ ID NO:1 and/or SEQ ID NO:3.

Another feature of this invention is the expression of the DNA sequencesdisclosed herein. As is well known in the art, DNA sequences may beexpressed by operatively linking them to an expression control sequencein an appropriate expression vector and employing that expression vectorto transform an appropriate unicellular host.

Such operative linking of a DNA sequence of this invention to anexpression control sequence, of course, includes, if not already part ofthe DNA sequence, the provision of an initiation codon, ATG, in thecorrect reading frame upstream of the DNA sequence.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol E1, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4;phage DNAs, e.g., the numerous derivatives of phage, e.g., NM989, andother phage DNA, e.g., M13 and filamentous single stranded phage DNA;yeast plasmids such as the 2 u plasmid or derivatives thereof; vectorsuseful in eukaryotic cells, such as vectors useful in insect ormammalian cells; vectors derived from combinations of plasmids and phageDNAs, such as plasmids that have been modified to employ phage DNA orother expression control sequences; and the like.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a DNA sequence operatively linked to it—may beused in these vectors to express the DNA sequences of this invention.Such useful expression control sequences include, for example, the earlyor late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lacsystem, the trp system, the TAC system, the TRC system, the LTR system,the major operator and promoter regions of phage, the control regions offd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), thepromoters of the yeast-mating factors, and other sequences known tocontrol the expression of genes of prokaryotic or eukaryotic cells ortheir viruses, and various combinations thereof.

A wide variety of unicellular host cells are also useful in expressingthe DNA sequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animalcells, such as CHO, YB/20, NSO, SP2/0, R1.1, B-W and L-M cells, AfricanGreen Monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10),insect cells (e.g., Sf9), and human cells and plant cells in tissueculture.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA sequences ofthis invention. Neither will all hosts function equally well with thesame expression system. However, one skilled in the art will be able toselect the proper vectors, expression control sequences, and hostswithout undue experimentation to accomplish the desired expressionwithout departing from the scope of this invention. For example, inselecting a vector, the host must be considered because the vector mustfunction in it. The vector's copy number, the ability to control thatcopy number, and the expression of any other proteins encoded by thevector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular DNA sequence or gene to be expressed, particularly asregards potential secondary structures. Suitable unicellular hosts willbe selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins correctly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the DNA sequences to beexpressed, and the ease of purification of the expression products.

Considering these and other factors a person skilled in the art will beable to construct a variety of vector/expression control sequence/hostcombinations that will express the DNA sequences of this invention onfermentation or in large scale animal culture.

It is further intended that specific binding member analogs may beprepared from nucleotide sequences of the protein complex/subunitderived within the scope of the present invention. Analogs, such asfragments, may be produced, for example, by pepsin digestion of specificbinding member material. Other analogs, such as muteins, can be producedby standard site-directed mutagenesis of specific binding member codingsequences. Analogs exhibiting “specific binding member activity” such assmall molecules, whether functioning as promoters or inhibitors, may beidentified by known in vivo and/or in vitro assays.

As mentioned above, a DNA sequence encoding a specific binding membercan be prepared synthetically rather than cloned. The DNA sequence canbe designed with the appropriate codons for the specific binding memberamino acid sequence. In general, one will select preferred codons forthe intended host if the sequence will be used for expression. Thecomplete sequence is assembled from overlapping oligonucleotidesprepared by standard methods and assembled into a complete codingsequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al.,Science, 223:1299 (1984); Jay et al., J. Biol. Chem., 259:6311 (1984).

Synthetic DNA sequences allow convenient construction of genes whichwill express specific binding member analogs or “muteins”.Alternatively, DNA encoding muteins can be made by site-directedmutagenesis of native specific binding member genes or cDNAs, andmuteins can be made directly using conventional polypeptide synthesis.

A general method for site-specific incorporation of unnatural aminoacids into proteins is described in Christopher J. Noren, Spencer J.Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, Science,244:182-188 (April 1989). This method may be used to create analogs withunnatural amino acids.

The present invention extends to the preparation of antisenseoligonucleotides and ribozymes that may be used to interfere with theexpression of the EGFR at the translational level. This approachutilizes antisense nucleic acid and ribozymes to block translation of aspecific mRNA, either by masking that mRNA with an antisense nucleicacid or cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule. (See Weintraub, 1990;Marcus-Sekura, 1988.) In the cell, they hybridize to that mRNA, forminga double stranded molecule. The cell does not translate an mRNA in thisdouble-stranded form. Therefore, antisense nucleic acids interfere withthe expression of mRNA into protein. Oligomers of about fifteennucleotides and molecules that hybridize to the AUG initiation codonwill be particularly efficient, since they are easy to synthesize andare likely to pose fewer problems than larger molecules when introducingthem into—producing cells. Antisense methods have been used to inhibitthe expression of many genes in vitro (Marcus-Sekura, 1988; Hambor etal., 1988).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single stranded RNA molecules in a manner somewhatanalogous to DNA restriction endonucleases. Ribozymes were discoveredfrom the observation that certain mRNAs have the ability to excise theirown introns. By modifying the nucleotide sequence of these RNAs,researchers have been able to engineer molecules that recognize specificnucleotide sequences in an RNA molecule and cleave it (Cech, 1988).Because they are sequence-specific, only mRNAs with particular sequencesare inactivated.

Investigators have identified two types of ribozymes, Tetrahymena-typeand “hammerhead”-type. (Hasselhoff and Gerlach, 1988) Tetrahymena-typeribozymes recognize four-base sequences, while “hammerhead”-typerecognize eleven- to eighteen-base sequences. The longer the recognitionsequence, the more likely it is to occur exclusively in the target mRNAspecies. Therefore, hammerhead-type ribozymes are preferable toTetrahymena-type ribozymes for inactivating a specific mRNA species, andeighteen base recognition sequences are preferable to shorterrecognition sequences.

The DNA sequences described herein may thus be used to prepare antisensemolecules against, and ribozymes that cleave mRNAs for EFGRs and theirligands.

The invention may be better understood by reference to the followingnon-limiting Examples, which are provided as exemplary of the invention.The following examples are presented in order to more fully illustratethe preferred embodiments of the invention and should in no way beconstrued, however, as limiting the broad scope of the invention.

EXAMPLE 1 Isolation of Antibodies

Materials

Cell Lines

For immunization and specificity analyses, several cell lines, native ortransfected with either the normal, wild type or “wtEGFR” gene or theΔEGFR gene carrying the Δ2-7 deletion mutation were used: Murinefibroblast cell line NR6, NR6_(ΔEGFR) (transfected with ΔEGFR) andNR6_(wtEGFR) (transfected with wtEGFR), human glioblastoma cell lineU87MG (expressing low levels of endogenous wtEGFR), U87MG_(wtEGFR)(transfected with wtEGFR), U87MG_(ΔEGFR) (transfected with ΔEGFR), andhuman squamous cell carcinoma cell line A431 (expressing high levels ofwtEGFR)[38]. Cell lines and transfections were described previously(Nishikawa R., et al. (1994) Proc. Natl. Acad. Sci. 91(16):7727-7731).

The U87MG astrocytoma cell line (20) (ATCC Accession No. HTB-14), whichendogenously expresses low levels of the wt EGFR, was infected with aretrovirus containing the de2-7 EGFR to produce the U87MG.Δ2-7 cell line(10). The transfected cell line U87MG.wtEGFR was produced as describedin Nagane et al 1996 (Cancer Res., 56: 5079-5086). Whereas U87MG cellsexpress approximately 1×10⁵ EGFR, U87MG.wtEGFR cells expressapproximately 1×10⁶ EGFR, and thus mimic the situation seen with geneamplification.

Human squamous carcinoma A431 cells were obtained from ATCC (Rockville,Md.). All cell lines were cultured in DMEM/F-12 with GlutaMAX™ (LifeTechnologies, Melbourne, Australia) supplemented with 10% FCS(CSL,Melbourne, Australia).

Reagents

Biotinylated unique junctional peptides (Biotin-LEEKKGNYVVTDH (SEQ IDNO:5) and LEEKKGNYVVTDH-Biotin (SEQ ID NO:6)) from de2-7 EGFR weresynthesized by standard Fmoc chemistry and purity (>96%) determined byreverse phase HPLC and mass spectral analysis (Auspep, Melbourne,Australia).

Antibodies Used in Studies

In order to compare our findings with other reagents, additional mAbswere included in our studies. These reagents were mAb 528 to the wtEGFR(Sato, J. D. et al. (1983) Mol. Biol. Med. 1(5):511-529) and DH8.3,which was generated against a synthetic peptide spanning the junctionalsequence of the 42-7 EGFR deletion mutation. The DH8.3 antibody (IgG1)has been described previously (Hills et al, 1995, Int. J. Cancer 63(4);537-543) and was obtained following immunization of mice with the uniquejunctional peptide found in de2-7 EGFR (16). The 528 antibody, whichrecognizes both de2-7 and wild type EGFR, has been described previously(21) and was produced in the Biological Production Facility (LudwigInstitute for Cancer Research, Melbourne) using a hybridoma obtainedfrom ATCC HB-8509. SC-03 is an affinity purified rabbit polyclonalantibody raised against a carboxy terminal peptide of the EGFR (SantaCruz Biotechnology Inc.).

Generation of Monoclonal Antibodies

The murine fibroblast line NR6_(ΔEGFR) was used as immunogen. Mousehybridomas were generated by immunizing BALB/c mice five timessubcutaneously at 2- to 3-week intervals, with 5×10⁵-2×10⁶ cells inadjuvant. Complete Freund's adjuvant was used for the first injection.Thereafter, incomplete Freund's adjuvant (Difco) was used. Spleen cellsfrom immunized mice were fused with mouse myeloma cell line SP2/0.Supernatants of newly generated clones were screened in hemadsorptionassays for reactivity with cell line NR6, NR6_(wtEGFR), and NR6_(ΔEGFR)and then analyzed by hemadsorption assays with human glioblastoma celllines U87MG, U87MG_(wtEGFR), and U87MG_(ΔEGFR). Selected hybridomasupernatants were subsequently tested by western blotting and furtheranalyzed by immunohistochemistry. Newly generated mAbs showing theexpected reactivity pattern were purified.

Five hybridomas were established and three, clones 124 (IgG2a), 806(IgG2b) (hybridoma 806, which produced mAb806, was deposited with theAmerican Type Culture Collection (1801 University Blvd., Manassas, Va.20110) as ATCC Deposit Number PTA-3858 on Nov. 4, 2001 under theprovisions of the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms), and 1133 (IgG2a) were selected forfurther characterization based on high titer with NR6_(ΔEGFR) and lowbackground on NR6 and NR6_(wtEGFR) cells in the hemagglutination assay.In a subsequent hemagglutination analysis, these antibodies showed noreactivity (undiluted supernatant <10%) with the native humanglioblastoma cell line U87MG and U87MG_(wtEGFR), but were stronglyreactive with U87MG_(ΔEGFR); less reactivity was seen with A431. Bycontrast, in FACS analysis, 806 was unreactive with native U87MG andintensively stained U87MG_(ΔEGFR) and to a lesser degree U87MG_(wtEGFR)indicating binding of 806 to both, ΔEGFR and wtEGFR (see below).

In Western blot assays, mAbs 124, 806 and 1133 were then analyzed forreactivity with wtEGFR and ΔEGFR. Detergent lysates were extracted fromNR6_(ΔEGFR), U87MG_(ΔEGFR) as well as from A431. All three mAbs showed asimilar reactivity pattern with cell lysates staining both the wtEGFR(170 kDa) and ΔEGFR protein (140 kDa). As a reference reagent, mAb R.Iknown to be reactive with the wtEGFR (Waterfield M. D. et al. (1982) J.Cell Biochem. 20(2):149-161) was used instead of mAb 528, which is knownto be non-reactive in western blot analysis. Mab R.I showed reactivitywith wt and ΔEGFR. All three newly generated clones showed reactivitywith ΔEGFR and less intense with wtEGFR. DH8.3 was solely positive inthe lysate of U87MG_(ΔEGFR) and NR6_(ΔEGFR).

The immunohistochemical analysis of clones 124, 806, and 1133 as well asmAb 528 and mAb DH8.3 on xenograft tumors U87MG, U87MG_(ΔEGFR), and A431are shown in Table 1. All mAbs showed strong staining of xenograftU87MG_(ΔEGFR). Only mAb 528 showed weak reactivity in the native U87MGxenograft. In A431 xenografts, mAb 528 showed strong homogeneousreactivity. MAbs 124, 806, and 1133 revealed reactivity with mostly thebasally located cells of the squamous cell carcinoma of A431 and did notreact with the upper cell layers or the keratinizing component. DH8.3was negative in A431 xenografts.

TABLE 1 Immunohistochemical Analysis of Antibodies 528, DH8.3, and 124,806 and 1133 xenograft xenograft U87MG Mab ΔU87MG_(ΔEGFR) xenograft A431(native) 528 pos. pos. pos. (focal staining) mAb-124 pos. pos.(predominantly basal − cells) mAb-806 pos. pos. (predominantly basal −cells) mAb-1133 pos. pos. (predominantly basal − cells) DH8.3 pos. − −minor stromal staining due to detection of endogenous mouse antibodies

EXAMPLE 2 Binding of Antibodies to Cell Lines by FACS

In order to determine the specificity of mAb 806, its binding to U87MG,U87MG.Δ2-7 and U87MG.wtEGFR cells was analyzed by flow activated cellsorting (FACS). Briefly, cells were labelled with the relevant antibody(10 μg/ml) followed by fluorescein-conjugated goat anti-mouse IgG (1:100dilution; Calbiochem San Diego, USA). FACS data was obtained on aCoulter Epics Elite™ ESP by observing a minimum of 5,000 events andanalysed using EXPO™ (version 2) for Windows. An irrelevant IgG2b wasincluded as an isotype control for mAb 806 and the 528 antibody wasincluded as it recognizes both the de2-7 and wt EGFR.

Only the 528 antibody was able to stain the parental U87MG cell line(FIG. 1) consistent with previous reports demonstrating that these cellsexpress the wt EGFR (Nishikawa et al, 1994). MAb 806 and DH8.3 hadbinding levels similar to the control antibody, clearly demonstratingthat they are unable to bind the wt receptor (FIG. 1). Binding of theisotype control antibody to U87MG.Δ2-7 and U87MG.wtEGFR cells wassimilar as that observed for the U87MG cells.

MAb 806 stained U87MG.Δ2-7 and U87MG.wtEGFR cells, indicating that mAb806 specifically recognizes the de2-7 EGFR and amplified EGFR (FIG. 1).DH8.3 antibody stained U87MG.Δ2-7 cells, confirming that DH8.3 antibodyspecifically recognizes the de2-7 EGFR (FIG. 1). As expected, the 528antibody stained both the U87MG.Δ2-7 and U87MG.wtEGFR cell lines (FIG.1). As expected, the 528 antibody stained U87MG.Δ2-7 with a higherintensity than the parental cell as it binds both the de2-7 and wildtype receptors that are co-expressed in these cells (FIG. 1). Similarresults were obtained using a protein A mixed hemadsorption whichdetects surface bound IgG by appearance of Protein A coated with humanred blood cells (group O) to target cells. Monoclonal antibody 806 wasreactive with U87MG.Δ2-7 cells but showed no significant reactivity(undiluted supernatant less than 10%) with U87MG expressing wild typeEGFR.

EXAMPLE 3 Binding of Antibodies in Assays

To further characterize the specificity of mAb 806 and the DH8.3antibody, their binding was examined by ELISA. Two types of ELISA wereused to determine the specificity of the antibodies. In the first assay,plates were coated with sEGFR (10 μg/ml in 0.1 M carbonate buffer pH9.2) for 2 h and then blocked with 2% human serum albumin (HSA) in PBS.sEGFR is the recombinant extracellular domain (amino acids 1-621) of thewild type EGFR), and was produced as previously described (22).Antibodies were added to wells in triplicate at increasing concentrationin 2% HSA in phosphate-buffered saline (PBS). Bound antibody wasdetected by horseradish peroxidase conjugated sheep anti-mouse IgG(Silenus, Melbourne, Australia) using ABTS (Sigma, Sydney, Australia) asa substrate and the absorbance measured at 405 nm.

Both mAb 806 and the 528 antibody displayed dose-dependent andsaturating binding curves to immobilized wild type sEGFR (FIG. 2A). Asthe unique junctional peptide found in the de2-7 EGFR is not containedwithin the sEGFR, mAb 806 must be binding to an epitope located withinthe wild type EGFR sequence. The binding of the 528 antibody was lowerthan that observed for mAb 806. As expected the DH8.3 antibody did notbind the wild type sEGFR even at concentrations up to 10 μg/ml (FIG.2A). While sEGFR in solution inhibited the binding of the 528 antibodyto immobilized sEGFR in a dose-dependent fashion, it was unable toinhibit the binding of mAb 806 (FIG. 2B). This suggests that mAb 806 canonly bind wild type EGFR once immobilized on ELISA plates, a processthat may induce conformational changes. Similar results were observedusing a BIAcore™ whereby mAb 806 bound immobilized sEGFR but immobilizedmAb 806 was not able to bind sEGFR in solution (data not shown). TheDH8.3 antibody exhibited dose-dependent and saturable binding to theunique de2-7 EGFR peptide (FIG. 2C).

In the second assay, the biotinylated de2-7 specific peptide(Biotin-LEEKKGNYVVTDH (SEQ ID NO:5)) was bound to ELISA plates precoatedwith streptavidin (Pierce, Rockford, Ill.). Antibodies were bound anddetected as in the first assay. Neither mAb 806 nor the 528 antibodybound to the peptide, even at concentrations higher than those used toobtain saturation binding of DH8.3, further indicating that mAb 806 doesnot recognize an epitope determinant within this peptide.

To further demonstrate that mAb 806 recognizes an epitope distinct fromthe junction peptide, additional experiments were performed. C-terminalbiotinylated de2-7 peptide (LEEKKGNYVVTDH-Biotin (SEQ ID NO:6)) wasutilized in studies with mAb806 and mAb L8A4, generated against thede2-7 peptide (Reist, C J et al (1995) Cancer Res. 55(19):4375-4382;Foulon C F et al. (2000) Cancer Res. 60(16):4453-4460).

Reagents Used in Peptide Studies:

Junction Peptide: (SEQ ID NO: 13) LEEKKGNYVVTDH-OH(Biosource, Camarillo, CA). Peptide C: (SEQ ID NO: 14)LEEKKGNYVVTDH(K-Biot)-OH (Biosource, Camarillo, CA).

-   sEGFR: CHO-cell-derived recombinant soluble extracellular domain (aa    1-621) of the wild type EGFR (LICR Melbourne).-   mAb 806: mouse monoclonal antibody, IgG_(2b) (LICR NYB).-   mAb L8A4: mouse monoclonal antibody, IgG₁ (Duke University).-   IgG₁ isotype control mAb.-   IgG_(2b) isotype control mAb.

Peptide C was immobilized on a Streptavidin microsensor chip at asurface density of 350RU (+/−30RU). Serial dilutions of mAbs were testedfor reactivity with the peptide. Blocking experiments usingnon-biotinylated peptide were performed to assess specificity.

MAb L8A4 showed strong reactivity with Peptide C even at low antibodyconcentrations (6.25 nM) (FIG. 2.D). mAb 806 did not show detectablespecific reactivity with peptide C up to antibody concentrations of 100nM (highest concentration tested) (FIGS. 2D AND 2E). It was expectedthat mAb L8A4 would react with Peptide C because the peptide was used asthe immunogen in the generation of mAb L8A4. Addition of the JunctionPeptide (non-biotinylated, 50 μg/ml) completely blocks the reactivity ofmAb L8A4 with Peptide C, confirming the antibody's specificity for thejunction peptide epitope.

In a second set of BIACORE™ experiments, sEGFR was immobilized on a CMmicrosensor chip at a surface density of ˜4000RU. Serial dilutions ofmAbs were tested for reactivity with sEGFR.

MAb 806 was strongly reactive with denaturated sEGFR while mAb L8A4 didnot react with denaturated sEGFR. Reactivity of mAb 806 with denaturatedsEGFR decreases with decreasing antibody concentrations. It was expectedthat mAb L8A4 does not react with sEGFR because mAb L8A4 was generatedusing the junction peptide as the immunogen and sEGFR does not containthe junction peptide.

Dot-blot immune stain experiments were also performed. Serial dilutionsof peptide were spotted in 0.5 μl onto a PVDF or nitrocellulosemembranes. Membranes were blocked with 2% BSA in PBS, and then probedwith 806, L8A4, DH8.3 and control antibodies. Antibodies L8A4 and DH8.3bound to peptide on the membranes (data not shown). Mab806 did not bindpeptide at concentrations where L8A4 clearly showed binding (data notshown). Control antibodies were also negative for peptide binding.

mAb 806 bound to the wtEGFR in cell lysates following immunoblotting(results not shown). This is different from the results obtained withDH8.3 antibody, which reacted with de2-7 EGFR but not wtEGFR. Thus, mAb806 can recognize the wtEGFR following denaturation but not when thereceptor is in its natural state on the cell surface.

EXAMPLE 4 Scatchard Analysis

A Scatchard analysis using U87MG.Δ2-7 cells was performed followingcorrection for immunoreactivity in order to determine the relativeaffinity of each antibody. Antibodies were labelled with ¹²⁵I (Amrad,Melbourne, Australia) by the chloramine T method and immunoreactivitydetermined by Lindmo assay (23). All binding assays were performed in 1%HSA/PBS on 1−2×10⁶ live U87MG.Δ2-7 or A431 cells for 90 min at 4° C.with gentle rotation. A set concentration of 10 ng/ml ¹²⁵I-labeledantibody was used in the presence of increasing concentrations of theappropriate unlabeled antibody. Non-specific binding was determined inthe presence of 10,000-fold excess of unlabeled antibody. After theincubation was completed, cells were washed and counted for bound¹²⁵I-labeled antibody using a COBRA II gamma counter (Packard InstrumentCompany, Meriden, USA).

Both mAb 806 and the DH8.3 antibody retained high immunoreactivity wheniodinated and was typically greater than 90% for mAb 806 and 45-50% forthe DH8.3 antibody. mAb 806 had an affinity for the de2-7 EGFR receptorof 1.1×10⁹ M⁻¹ whereas the affinity of DH8.3 was some 10-fold lower at1.0×10⁸ M⁻¹. Neither ¹²⁵I-radiolabeled mAb 806 nor the ¹²⁵I-radiolabeledDH8.3 antibody bound to parental U87MG cells. mAb 806 recognized anaverage of 2.4×10⁵ binding sites per cell with the DH8.3 antibodybinding an average of 5.2×10⁵ sites. Thus, there was not only goodagreement in receptor number between the antibodies, but also with aprevious report showing 2.5×10⁵ de2-7 receptors per cell as measured bya different de2-7 EGFR specific antibody on the same cell line (25).

EXAMPLE 5 Internalization of Antibodies by U87MG.Δ2-7 Cells

The rate of antibody internalization following binding to a target cellinfluences both its tumor targeting properties and therapeutic options.Consequently, the inventors examined the internalization of mAb 806 andthe DH8.3 antibody following binding to U87MG.Δ2-7 cells by FACS.U87MG.Δ2-7 cells were incubated with either mAb 806 or the DH8.3antibody (10 μg/ml) for 1 h in DMEM at 4° C. After washing, cells weretransferred to DMEM pre-warmed to 37° C. and aliquots taken at varioustime points following incubation at 37° C. Internalization was stoppedby immediately washing aliquots in ice-cold wash buffer (1% HSA/PBS). Atthe completion of the time course cells were stained by FACS asdescribed above. Percentage internalization was calculated by comparingsurface antibody staining at various time points to zero time using theformula: percent antibody internalized=(mean fluorescence attime×background fluorescence)/(mean fluorescence at time 0−backgroundfluorescence)×100. This method was validated in one assay using aniodinated antibody (mAb 806) to measure internalization as previouslydescribed (24). Differences in internalization rate at different timepoints were compared using Student's t-test.

Both antibodies showed relatively rapid internalization reachingsteady-state levels at 10 min for mAb 806 and 30 min for DH8.3 (FIG. 3).Internalization of DH8.3 was significantly higher both in terms of rate(80.5% of DH8.3 internalized at 10 min compared to 36.8% for mAb 806,p<0.01) and total amount internalized at 60 min (93.5% versus 30.4%,p<0.001). mAb 806 showed slightly lower levels of internalization at 30and 60 min compared to 20 min in all 4 assays performed (FIG. 3). Thisresult was also confirmed using an internalization assay based oniodinated mAb 806.

EXAMPLE 6 Electron Microscopy Analysis of Antibody Internalization

Given the above noted difference in internalization rates between theantibodies, a detailed analysis of antibody intracellular traffickingwas performed using electron microscopy.

U87MG.Δ2-7 cells were grown on gelatin coated chamber slides (Nunc,Naperville, Ill.) to 80% confluence and then washed with ice cold DMEM.Cells were then incubated with mAb 806 or the DH8.3 antibody in DMEM for45 min at 4° C. After washing, cells were incubated for a further 30 minwith gold-conjugated (20 nm particles) anti-mouse IgG (BBInternational,Cardiff, UK) at 4° C. Following a further wash, pre-warmed DMEM/10% FCSwas added to the cells, which were incubated at 37° C. for various timesfrom 1-60 min. Internalization of the antibody was stopped by ice-coldmedia and cells fixed with 2.5% glutaraldehyde in PBS/0.1% HSA and thenpost-fixed in 2.5% osmium tetroxide. After dehydration through a gradedseries of acetone, samples were embedded in Epon/ARALDITE® resin, cut asultra-thin sections with a Reichert Ultracut-S microtome (Leica) andcollected on nickel grids. The sections were stained with uranyl acetateand lead citrate before being viewed on a Philips CM12 transmissionelectron microscope at 80 kV. Statistical analysis of gold grainscontained within coated pits was performed using a Chi-square test.

While the DH8.3 antibody was internalized predominantly via coated-pits,mAb 806 appeared to be internalized by macropinocytosis (FIG. 19). Infact, a detailed analysis of 32 coated pits formed in cells incubatedwith mAb 806 revealed that none of them contained antibody. In contrast,around 20% of all coated-pits from cells incubated with DH8.3 werepositive for antibody, with a number containing multiple gold grains. Astatistical analysis of the total number of gold grains contained withincoated-pits found that the difference was highly significant (p<0.01).After 20-30 min both antibodies could be seen in structures thatmorphologically resemble lysosomes. The presence of cellular debriswithin these structures was also consistent with their lysosome nature.

EXAMPLE 7 Biodistribution of Antibodies in Tumor Bearing Nude Mice

The biodistribution of mAb 806 and the DH8.3 antibody was compared innude mice containing U87MG xenografts on one side and U87MG.Δ2-7xenografts on the other. A relatively short time period was chosen forthis study as a previous report demonstrated that the DH8.3 antibodyshows peak levels of tumor targeting between 4-24 h (16).

Tumor xenografts were established in nude BALB/c mice by s.c. injectionof 3×10⁶ U87MG, U87MG.Δ2-7 or A431 cells. de2-7 EGFR expression inU87MG.Δ2-7 xenografts remained stable throughout the period ofbiodistribution. Also, A431 cells retained their mAb 806 reactivity whengrown as tumor xenografts as determined by immunohistochemistry (datanot shown). U87MG or A431 cells were injected on one side 7-10 daysbefore U87MG.Δ2-7 cells were injected on the other side because of thefaster growth rate observed for de2-7 EGFR expressing xenografts.Antibodies were radiolabeled and assessed for immunoreactivity asdescribed above and were injected into mice by the retro-orbital routewhen tumors were 100-200 mg in weight. Each mouse received two differentantibodies (2 μg per antibody): 2 μCi of ¹²⁵I-labeled mAb 806 and 2 μCiof ¹³¹I-labelled DH8.3 or 528. Unless indicated, groups of 5 mice weresacrificed at various time points post-injection and blood obtained bycardiac puncture. The tumors, liver, spleen, kidneys and lungs wereobtained by dissection. All tissues were weighed and assayed for ¹²⁵Iand ¹³¹I activity using a dual-channel counting Window. Data wasexpressed for each antibody as % ID/g tumor determined by comparison toinjected dose standards or converted into tumor to blood/liver ratios(i.e. % ID/g tumor divided by % ID/g blood or liver). Differencesbetween groups were analysed by Student's t-test.

In terms of % ID/g tumor, mAb 806 reached its peak level in U87MG.Δ2-7xenografts of 18.6% m/g tumor at 8 h (FIG. 4A), considerably higher thanany other tissue except blood. While DH8.3 also showed peak tumor levelsat 8 h, the level was a statistically (p<0.001) lower 8.8% m/g tumorcompared to mAb 806FIG. 4B). Levels of both antibodies slowly declinedat 24 and 48 h. Neither antibody showed specific targeting of U87MGparental xenografts (FIGS. 4A-B). With regards to tumor to blood/liverratios, mAb 806 showed the highest ratio at 24 h for both blood (ratioof 1.3) and liver (ratio of 6.1) (FIGS. 5 A-B). The DH8.3 antibody hadits highest ratio in blood at 8 h (ratio of 0.38) and at 24 h in liver(ratio of 1.5) (FIGS. 5A-B), both of which are considerably lower thanthe values obtained for mAb 806.

As described above, levels of mAb 806 in the tumor peaked at 8 hours.While this peak is relatively early compared to many tumor-targetingantibodies, it is completely consistent with other studies using de2-7EGFR specific antibodies which all show peaks at 4-24 hourspost-injection when using a similar dose of antibody (16, 25, 33).Indeed, unlike the earlier reports, the 8 h time point was included onthe assumption that antibody targeting would peak rapidly. The % ID/gtumor seen with mAb 806 was similar to that reported for other de2-7EGFR specific antibodies when using standard iodination techniques (16,24, 32). The reason for the early peak is probably two-fold. Firstly,tumors expressing the de2-7 EGFR, including the transfected U87MG cells,grow extremely rapidly as tumor xenografts. Thus, even during therelatively short period of time used in these biodistribution studies,the tumor size increases to such an extent (5-10 fold increase in massover 4 days) that the % ID/g tumor is reduced compared with slow growingtumors. Secondly, while internalization of mAb 806 was relatively slowcompared to DH8.3, it is still rapid with respect to many other tumorantibody/antigen systems. Internalized antibodies undergo rapidproteolysis with the degradation products being excreted from the cell(34). This process of internalization, degradation and excretion reducesthe amount of iodinated antibody retained within the cell. Consequently,internalizing antibodies display lower levels of targeting than theirnon-internalizing counterparts. The electron microscopy data reportedherein demonstrates that internalized mAb 806 is rapidly transported tolysosomes where rapid degradation presumably occurs. This observation isconsistent with the swift expulsion of iodine from the cell.

The previously described L8A4 monoclonal antibody directed to the uniquejunctional peptide found in the de2-7 EGFR, behaves in a similar fashionto mAb 806 (35). Using U87MG cells transfected with the de2-7 EGFR, thisantibody had a similar internalization rate (35% at 1 hour compared to30% at 1 hour for mAb 806) and displayed comparable in vivo targetingwhen using 3T3 fibroblasts transfected with de2-7 EGFR (peak of 24% ID/gtumor at 24 hours compared to 18% ID/g tumor at 8 hours for mAb 806)(25). Interestingly, in vivo retention of this antibody in tumorxenografts was enhanced when labeled with N-succinimidyl5-iodo-3-pyridine carboxylate (25). This labeled prosthetic group ispositively charged at lysosomal pH and thus has enhanced cellularretention (33). Enhanced retention is potentially useful whenconsidering an antibody for radioimmunotherapy and this method could beused to improve retention of iodinated mAb 806 or its fragments.

EXAMPLE 8 Binding of mAb 806 to Cells Containing Amplified EGFR

To examine if mAb 806 could recognize the EGFR expressed in cellscontaining an amplified receptor gene, its binding to A431 cells wasanalysed. As described previously, A431 cells are human squamouscarcinoma cells and express high levels of wtEGFR. Low, but highlyreproducible, binding of mAb 806 to A431 cells was observed by FACSanalysis (FIG. 6). The DH8.3 antibody did not bind A431 cells,indicating that the binding of mAb 806 was not the result of low levelde2-7 EGFR expression (FIG. 6). As expected, the anti-EGFR 528 antibodyshowed strong staining of A431 cells (FIG. 6). Given this result,binding of mAb 806 to A431 was characterized by Scatchard analysis.While the binding of iodinated mAb 806 was comparatively low, it waspossible to get consistent data for Scatchard. The average of suchexperiments gave a value for affinity of 9.5×10⁷ M⁻¹ with 2.4×10⁵receptors per cell. Thus the affinity for this receptor was some 10-foldlower than the affinity for the de2-7 EGFR. Furthermore, mAb 806 appearsto only recognize a small portion of EGFR found on the surface of A431cells. Using the 528 antibody, the inventors measured approximately2×10⁶ receptors per cell which is in agreement with numerous otherstudies(26).

Recognition of the wild type sEGFR by mAb 806 clearly requires somedenaturation of the receptor in order to expose the epitope. The extentof denaturation required is only slight as even absorption of the wildtype sEGFR on to a plastic surface induced robust binding of mAb 806 inELISA assays. As mAb 806 only bound approximately 10% of the EGFR on thesurface of A431 cells, it is tempting to speculate that this subset ofreceptors may have an altered conformation similar to that induced bythe de2-7 EGFR truncation. Indeed, the extremely high expression of theEGFR mediated by gene amplification in A431 cells, may cause somereceptors to be incorrectly processed leading to altered conformation.Interestingly, semi-quantitative immunoblotting of A431 cell lysateswith mAb 806 showed that it could recognize most of the A431 EGFreceptors following SDS-PAGE and western transfer. This result furthersupports the argument that mAb 806 is binding to a subset of receptorson the surface of A431 cells that have an altered conformation. Theseobservations in A431 cells are consistent with the immunohistochemistrydata demonstrating that mAb 806 binds gliomas containing amplificationof the EGFR gene. As mAb 806 binding was completely negative on parentalU87MG cells it would appear this phenomenon may be restricted to cellscontaining amplified EGFR although the level of “denatured” receptor onthe surface of U87MG cells may be below the level of detection. However,this would seem unlikely as iodinated mAb 806 did not bind to U87MG cellpellets containing up to 1×10⁷ cells.

EXAMPLE 9 In Vivo Targeting of A431 Cells by MAb 806

A second biodistribution study was performed with mAb 806 to determineif it could target A431 tumor xenografts. The study was conducted over alonger time course in order obtain more information regarding thetargeting of U87MG.Δ.2-7 xenografts by mAb 806, which were included inall mice as a positive control. In addition, the anti-EGFR 528 antibodywas included as a positive control for the A431 xenografts, since aprevious study demonstrated low but significant targeting of thisantibody to A431 cells grown in nude mice (21).

During the first 48 h, mAb 806 displayed almost identical targetingproperties as those observed in the initial experiments (FIG. 7Acompared with FIG. 4A). In terms of % ID/g tumor, levels of mAb 806 inU87MG.Δ2-7 xenografts slowly declined after 24 h but always remainedhigher than levels detected in normal tissue. Uptake in the A431xenografts was comparatively low, however there was a small increase in% ID/g tumor during the first 24 h not observed in normal tissues suchas liver, spleen, kidney and lung (FIG. 7A). Uptake of the 528 antibodywas very low in both xenografts when expressed as % ID/g tumor (FIG. 7B)partially due to the faster clearance of this antibody from the blood.In terms of tumor to blood ratio mAb 806 peaked at 72 h for U87MG.Δ2-7xenografts and 100 h for A431 xenografts (FIGS. 8A and 8B). While thetumor to blood ratio for mAb 806 never surpassed 1.0 with respect to theA431 tumor, it did increase throughout the entire time course (FIG. 8)and was higher than all other tissues examined (data not shown)indicating low levels of targeting.

The tumor to blood ratio for the 528 antibody showed a similar profileto mAb 806 although higher levels were noted in the A431 xenografts(FIGS. 8A and 8B). mAb 806 had a peak tumor to liver ratio in U87MG.Δ2-7xenografts of 7.6, at 72 h, clearly demonstrating preferential uptake inthese tumors compared to normal tissue (FIG. 8C). Other tumor to organratios for mAb 806 were similar to those observed in the liver (data notshown). The peak tumor to liver ratio for mAb 806 in A431 xenografts was2.0 at 100 h, again indicating a slight preferential uptake in tumorcompared with normal tissue (FIG. 8D).

EXAMPLE 10 Therapy Studies

The effects of mAb806 were assessed in two xenograft models of disease—apreventative model and an established tumor model.

Xenograft Models

Consistent with previous reports (Nishikawa et al, Proc. Natl. Acad.Sci. USA, 91(16); 7727-7731) U87MG cells transfected with de2-7 EGFRgrew more rapidly than parental cells and U87MG cells transfected withthe wt EGFR. Therefore, it was not possible to grow both cell types inthe same mice.

3×10⁶ tumor cells in 100 ml of PBS were inoculated subcutaneously. intoboth flanks of 4-6 week old female nude mice (Animal Research Centre,Western Australia, Australia). Therapeutic efficacy of mAb 806 wasinvestigated in both preventative and established tumor models. In thepreventative model, 5 mice with 2 xenografts each were treatedintraperitoneally. with either 1 or 0.1 mg of mAb 806 or vehicle (PBS)starting the day before tumor cell inoculation. Treatment was continuedfor a total of 6 doses, 3 times per week for 2 weeks. In the establishedmodel, treatment was started when tumors had reached a mean volume of65±6.42 mm³ (U87MG.A.2-7), 84±9.07 mm³ (U87MG), 73±7.5 mm³(U87MG.wtEGFR) or 201±19.09 mm³ (A431 tumors). Tumor volume in mm³ wasdetermined using the formula (length×width²)/2, where length was thelongest axis and width the measurement at right angles to the length(Scott et al, 2000). Data was expressed as mean tumor volume±S.E. foreach treatment group. Statistical analysis was performed at given timepoints using Student's t test. Animals were euthanased when thexenografts reached an approximate volume of 1.5 cm³ and the tumorsexcised for histological examination. This research project was approvedby the Animal Ethics Committee of the Austin and Repatriation MedicalCentre.

Histological Examination of Tumor Xenografts

Xenografts were excised and bisected. One half was fixed in 10%formalin/PBS before being embedded in paraffin. Four micron sectionswere then cut and stained with haematoxylin and eosin (H&E) for routinehistological examination. The other half was embedded in Tissue® O.C.T.™compound (Sakura Finetek, Torrance, Calif.), frozen in liquid nitrogenand stored at −80° C. Thin (5 micron) cryostat sections were cut andfixed in ice-cold acetone for 10 min followed by air drying for afurther 10 min. Sections were blocked in protein blocking reagent(Lipshaw Immunon, Pittsburgh U.S.A.) for 10 min and then incubated withbiotinylated primary antibody (1 mg/ml), for 30 min at room temperature(RT). All antibodies were biotinylated using the ECL™ proteinbiotinylation module (Amersham, Baulkham Hills, Australia), as per themanufactures instructions. After rinsing with PBS, sections wereincubated with a streptavidin horseradish peroxidase complex for afurther 30 min (Silenus, Melbourne, Australia). Following a final PBSwash the sections were exposed to 3-amino-9-ethylcarbozole (AEC)substrate (0.1 M acetic acid, 0.1 M sodium acetate, 0.02 M AEC (SigmaChemical Co., St Louis, Mo.)) in the presence of hydrogen peroxide for30 min. Sections were rinsed with water and counterstained withhematoxylin for 5 min and mounted.

Efficacy of mAb 806 in Preventative Model

MAb 806 was examined for efficacy against U87MG and U87MG.Δ2-7 tumors ina preventative xenograft model. Antibody or vehicle were administeredi.p. the day before tumor inoculation and was given 3 times per week for2 weeks. MAb 806 had no effect on the growth of parental U87MGxenografts, which express the wt EGFR, at a dose of 1 mg per injection(FIG. 9A). In contrast, mAb 806 significantly inhibited the growth ofU87MG.Δ2-7 xenografts in a dose dependent manner (FIG. 9B). At day 20,when control animals were sacrificed, the mean tumor volume was1637±178.98 mm³ for the control group, a statistically smaller 526±94.74mm³ for the 0.1 mg per injection group (p<0.0001) and 197±42.06 mm³ forthe 1 mg injection group (p<0.0001). Treatment groups were sacrificed atday 24 at which time the mean tumor volumes was 1287±243.03 mm³ for the0.1 mg treated group and 492±100.8 mm³ for the 1 mg group.

Efficacy of mAb 806 in Established Xenograft Model

Given the efficacy of mAb 806 in the preventative xenograft model, itsability to inhibit the growth of established tumor xenografts was thenexamined Antibody treatment was as described in the preventative modelexcept that it commenced when tumors had reached a mean tumor volume of65±6.42 mm³ for the U87MG.Δ2-7 xenografts and 84±9.07 mm³ for theparental U87MG xenografts. Once again, mAb 806 had no effect on thegrowth of parental U87MG xenografts at a dose of 1 mg per injection(FIG. 10A). In contrast, mAb 806 significantly inhibited the growth ofU87MG.Δ2-7 xenografts in a dose dependent manner (FIG. 10B). At day 17,one day before control animals were sacrificed, the mean tumor volumewas 935±215.04 mm³ for the control group, 386±57.51 mm³ for the 0.1 mgper injection group (p<0.01) and 217±58.17 mm³ for the 1 mg injectiongroup (p<0.002).

To examine whether the growth inhibition observed with mAb 806 wasrestricted to cell expressing de2-7 EGFR, its efficacy againstU87MG.wtEGFR tumor xenografts was examined in an established model.These cells serve as a model for tumors containing amplification of theEGFR gene without de2-7 EGFR expression. MAb 806 treatment commencedwhen tumors had reached a mean tumor volume of 73±7.5 mm³ MAb 806significantly inhibited the growth of established U87MG.wtEGFRxenografts when compared to control tumors treated with vehicle (FIG.10C). On the day control animals were sacrificed, the mean tumor volumewas 960±268.9 mm³ for the control group and 468±78.38 mm³ for the grouptreated with 1 mg injections (p<0.04).

Histological and Immunohistochemical Analysis of Established Tumors

To evaluate potential histological differences between mAb 806-treatedand control U87MG.Δ2-7 and U87MG.wtEGFR xenografts (collected at days 24and 42 respectively), formalin-fixed, paraffin embedded sections werestained with H&E.

Areas of necrosis were seen in sections from both U87MG.Δ2-7 (collected3 days after treatment finished), and U87MG.wtEGFR xenografts (collected9 days after treatment finished) treated with mAb 806. This result wasconsistently observed in a number of tumor xenografts (n=4). However,analysis of sections from xenografts treated with control did notdisplay the same areas of necrosis seen with mAb 806 treatment. Sectionsfrom mAb 806 or control treated U87MG xenografts were also stained withH&E and revealed no differences in cell viability between the twogroups, further supporting the hypothesis that mAb 806 binding inducesdecreased cell viability/necrosis within tumor xenografts.

An immunohistochemical analysis of U87MG, U87MG.Δ2-7 and U87MG.wtEGFRxenograft sections was performed to determine the levels of de2-7 and wtEGFR expression following mAb806 treatment. Sections were collected atdays 24 and 42 as above, and were immunostained with the 528 or 806antibodies. As expected the 528 antibody stained all xenograft sectionswith no obvious decrease in intensity between treated and controltumors. Staining of U87MG sections was undetectable with the mAb 806,however positive staining of U87MG.Δ2-7 and U87MG.wtEGFR xenograftsections was observed. There was no difference in mAb 806 stainingdensity between control and treated U87MG.Δ2-7 and U87MG.wtEGFRxenografts suggesting that antibody treatment does not down regulatede2-7 or wt EGFR expression.

Treatment of A431 Xenografts with mAb 806

To demonstrate that the anti-tumor effects of mAb 806 were notrestricted to U87MG cells, the antibody was administered to mice withA431 xenografts. These cells contain an amplified EGFR gene and expressapproximately 2×10⁶ receptors per cell. As described above, mAb 806binds about 10% of these EGFR and targets A431 xenografts. MAb 806significantly inhibited the growth of A431 xenografts when examined inthe previously described preventative xenograft model (FIG. 11A). At day13, when control animals were sacrificed, the mean tumor volume was1385±147.54 mm³ in the control group and 260±60.33 mm³ for the 1 mginjection treatment group (p<0.0001).

In a separate experiment, a dose of 0.1 mg mAb also significantlyinhibited the growth of A431 xenografts in a preventative model.

Given the efficacy of mAb 806 in the preventative A431 xenograft model,its ability to inhibit the growth of established tumor xenografts wasexamined Antibody treatment was as described in the preventative modelexcept it was not started until tumors had reached a mean tumor volumeof 201±19.09 mm³ MAb 806 significantly inhibited the growth ofestablished tumor xenografts (FIG. 11B). At day 13, when control animalswere sacrificed, the mean tumor volume was 1142±120.06 mm³ for thecontrol group and 451±65.58 mm³ for the 1 mg injection group (p<0.0001).

In summary, the therapy studies with mAb 806 described here clearlydemonstrated dose dependent inhibition of U87MG.Δ2-7 xenograft growth.In contrast, no inhibition of parental U87MG xenografts was observeddespite the fact they continue to express the wt EGFR in vivo. MAb 806not only significantly reduced xenograft volume, it also inducedsignificant necrosis within the tumor. This is the first report showingthe successful therapeutic use of such an antibody in vivo against ahuman de2-7 EGFR expressing glioma xenografts.

Gene amplification of the EGFR has been reported in a number ofdifferent tumors and is observed in approximately 50% of gliomas(Voldberg et al, 1997). It has been proposed that the subsequent EGFRover-expression mediated by receptor gene amplification may confer agrowth advantage by increasing intracellular signalling and cell growth(Filmus et al, 1987). The U87MG cell line was transfected with the wtEGFR in order to produce a glioma cell that mimics the process of EGFRgene amplification. Treatment of established U87MG.wtEGFR xenograftswith mAb 806 resulted in significant growth inhibition. Thus, mAb 806also mediates in vivo anti-tumor activity against cells containingamplification of the EGFR gene. Interestingly, mAb 806 inhibition ofU87MG.wtEGFR xenografts appears to be less effective than that observedwith U87MG.Δ2-7 tumors. This probably reflects the fact that mAb 806 hasa lower affinity for the amplified EGFR and only binds a smallproportion of receptors expressed on the cell surface. However, itshould be noted that despite the small effect on U87MG.wtEGFR xenograftvolumes, mAb 806 treatment produced large areas of necrosis within thesexenografts. To rule out the possibility that mAb 806 only mediatesinhibition of the U87MG derived cell lines we tested its efficacyagainst A431 xenografts. This squamous cell carcinoma derived cell linecontains significant EGFR gene amplification which is retained both invitro and in vivo. Treatment of A431 xenografts with mAb 806 producedsignificant growth inhibition in both a preventative and establishedmodel, indicating the anti-tumor effects of mAb 806 are not restrictedto transfected U87MG cell lines.

EXAMPLE 11 Combination Therapy Treatment of A431 Xenografts with MAb806and AG1478

The anti-tumor effects of mAb 806 combined with AG1478 was tested inmice with A431 xenografts. AG1478(4-(3-Chloroanilino)-6,7-dimethoxyquinazoline) is a potent and selectiveinhibitor of the EGFR kinase versus HER2-neu and platelet-derived growthfactor receptor kinase (Calbiochem Cat. No. 658552). Three controls wereincluded: treatment with vehicle only, vehicle+mAb806 only andvehicle+AG1478 only. The results are illustrated in FIG. 12. 0.1 mgmAb806 was administered at 1 day prior to xenograft and 1, 3, 6, 8 and10 days post xenograft. 400 μg AG1478 was administered at 0, 2, 4, 7, 9,and 11 days post xenograft.

Both AG1478 and mAb806, when administered alone produced a significantreduction of tumor volume. However, in combination, the reduction oftumor volume was greatly enhanced.

In addition, the binding of mAb806 to EGFR of A431 cells was evaluatedin the absence and presence of AG1478. Cells were placed in serum freemedia overnight, then treated with AG1478 for 10 min at 37° C., washedtwice in PBS then lysed in 1% Triton and lysates prepared. The lysateswere prepared as described in Example 20 herein. Lysate was thenassessed for 806 reactivity by an ELISA is a modified version of anassay described by Schooler and Wiley, Analytical Biochemistry 277,135-142 (2000). Plates were coated with 10 μg/ml of mAb 806 in PBS/EDTAovernight at room temperature and then washed twice. Plates were thenblocked with 10% serum albumin/PBS for 2 hours at 37° C. and washedtwice. A 1:20 cell lysate was added in 10% serum albumin/PBS for 1 hourat 37° C., then washed four times. Anti-EGFR(SC-03 (Santa CruzBiotechnology Inc.)) in 10% serum albumin/PBS was reacted 90 min at roomtemperature, the plate washed four times, and anti-rabbit-HRP (1:2000 iffrom Silenus) in 10% serum albumin/PBS was added for 90 min at roomtemperature, washed four timed, and color developed using ABTS as asubstrate. It was found that mAb806 binding is significantly increasedin the presence of increasing amounts of AG1478 (FIG. 13).

EXAMPLE 12 Immunoreactivity in Human Glioblastomas Pre-Typed for EGFRStatus

Given the high incidence of EGFR expression, amplification and mutationin glioblastomas, a detailed immunohistochemical study was performed inorder to assess the specificity of 806 in tumors other than xenografts.A panel of 16 glioblastomas was analyzed by immunohistochemistry. Thispanel of 16 glioblastomas was pre-typed by RT-PCR for the presence ofamplified wild-type EGFR and de2-7 EGFR expression. Six of these tumorsexpressed only the wt EGFR transcript, 10 had wtEGFR gene amplificationwith 5 of these showing wild-type EGFR transcripts only, and 5 bothwild-type EGFR and de2-7 gene transcript. Immunohistochemical analysiswas performed using 5 mm sections of fresh frozen tissue applied tohistology slides and fixed for 10 minutes in cold acetone. Bound primaryantibody was detected with biotinylated horse anti-mouse antibodyfollowed by an avidin-biotin-complex reaction. Diaminobenzidinetetrahydrochloride (DAB) was used as chromogen. The extent of theimmunohistochemical reactivity in tissues was estimated by lightmicroscopy and graded according to the number of immunoreactive cells in25% increments as follows:

-   -   Focal=less than 5%    -   +=5-25%    -   ++=25-50%    -   +++=50-75%    -   ++++=>75%

The 528 antibody showed intense reactivity in all tumors, while DH 8.3immunostaining was restricted to those tumors expressing the de2-7 EGFR(Table 2). Consistent with the previous observations in FACS androsetting assays, mAb 806 did not react with the glioblastomasexpressing the wtEGFR transcript from nonamplified EGFR genes (Table 2).This pattern of reactivity for mAb 806 is similar to that observed inthe xenograft studies and again suggests that this antibody recognizesthe de2-7 and amplified EGFR but not the wtEGFR when expressed on thecell surface.

TABLE 2 Immunoreactivity of MAbs 528, DH8.3 and 806 on glioblastomaspretyped for the presence of wild type EGFR and mutated de2-7 EGFR andfor their amplification status. de2-7 EGFR Amplification Expression 528DH8.3 806 No ++++ − − No ++++ − −* No ++++ − − No ++ − − No +++ − − No++++ − − Yes No ++++ − ++++ Yes No ++++ − + Yes No ++++ − +++ Yes No++++ − ++++ Yes No ++++ − +−++++ Yes Yes ++++ ++++ ++++ Yes Yes ++++++++ ++++ Yes Yes ++++ ++++ ++++ Yes Yes ++++ ++++ ++++ Yes Yes ++++ ++++ *focal staining

EXAMPLE 13 EGFR Immunoreactivity in Normal Tissue

In order to determine if the de2-7 EGFR is expressed in normal tissue,an immunohistochemical study with mAb 806 and DH8.3 was conducted in apanel of 25 tissues. There was no strong immunoreactivity with eithermAb 806 or DH8.3 in any tissue tested suggesting that the de2-7 EGFR isabsent in normal tissues (Table 3). There was some variable stainingpresent in tonsils with mAb 806 that was restricted to the basal celllayer of the epidermis and mucosal squamous cells of the epithelium. Inplacenta, occasional immunostaining of the trophoblast epithelium wasobserved. Interestingly, two tissues that express high endogenous levelsof wtEGFR, the liver and skin, failed to show any significant mAb 806reactivity. No reactivity was observed with the liver samples at all,and only weak and inconsistent focal reactivity was detectedoccasionally (in no more than 10% of all samples studied) in basalkeratinocytes in skin samples and in the squamous epithelium of thetonsil mucosa, further demonstrating that this antibody does not bindthe wtEGFR expressed on the surface of cells to any significant extent(Table 3). All tissues were positive for the wtEGFR as evidenced by theuniversal staining seen with the 528 antibody (Table 3).

TABLE 3 Reactivity of 582, DH8.3 and 806 on normal tissues. TISSUE 528DH8.3 806 Esophagus pos − − Stomach pos − − Duodenum pos − − Small pos −− intestine/duodenum Colon pos − − Liver pos − − Salivary glands(parotid) pos − − Kidney pos − − Urinary bladder pos − − Prostate pos −− Testis pos − − Uterus (cx/endom) pos −* − Fallopian tube pos − − Ovarypos − − Breast pos −* − Placenta pos − − Peripheral nerve pos − −Skeletal muscle pos − − Thyroid gland pos − − Lymph node pos − − Spleenpos − − Tonsil pos − − occ. weak reactivity of basal layer of squamousepithelium Heart pos − − Lung pos − − Skin pos − − occ. weak reactivityof basal layer of squamous epithelium *some stromal staining in varioustissue

EXAMPLE 14 EGFR Immunoreactivity in Various Tumors

The extent of de2-7 EGFR in other tumor types was examined using a panelof 12 different malignancies. The 528 antibody showed often homogeneousstaining in many tumors analysed except melanoma and seminoma. Whenpresent, DH8.3 immunoreactivity was restricted to the occasional focaltumor cell indicating there is little if any de2-7 EGFR expression intumors outside the brain using this detection system (Table 4). Therewas also focal staining of blood vessels and a varying diffuse stainingof connective tissue with the DH8.3 antibody in some tumors (Table 4).This staining was strongly dependent on antibody concentration used andwas considered nonspecific background reactivity. The mAb 806 showedpositive staining in 64% of head and neck tumors and 50% of lungcarcinomas (Table 4). There was little mAb 806 reactivity elsewhereexcept in urinary tumors that were positive in 30% of cases. Since thehead and neck and lung cancers were negative for the DH8.3 antibody thereactivity seen with the mAb in these tumors maybe associated with EGFRgene amplification.

TABLE 4 Monoclonal antibodies 528, DH8.3 and 806 on tumor panel. Tumor528 DH8.3 806 Malignant melanoma 0/10 0/10 0/10 metastases Urinarybladder (tcc, sqcc, 10/10 0/10* 3/10* adeno) (7x++++, 2x+++, 1x+)(2x++++, 1x++) Mammary gland 6/10 1/10 1/10 (3x++++, 3x++) (1x+) (foc)Head + neck cancer (sqcc) 11/11 0/11* 7/11 1x+++-10x++++) (3x++++,3x+++, 1x+) Lung (sqcc, adeno, neuroend) 12/12 0/12* 6/12 10x++++-1x+++)(3x++++ 3x+++) Leiomyosarcoma 5/5 0/5 0/5 (4x++++, 1x+) Liposarcoma 5/50/5 0/5* (2x + 3x +++) Synovial sarcoma 4/5* 0/5 0/5* (4x ++++) MfhMalignant fibrous histiocytoma 4/5* 0/5* 0/5* Colonic carcinoma 10/100/10* 0/10 (9x++++, 1x+) Seminoma 1/10* 1/10* 0/10 Ovary(serous-papillary) 4/5 0/5* 0/5 (3x++++, 1x+) *focal staining

EXAMPLE 15 Immunoreactivity in Human Glioblastomas Unselected for EGFRStatus

In order to confirm the unique specificity and to evaluate thereactivity of mAb 806, it was compared to the 528 and DH8.3 antibodiesin a panel of 46 glioblastomas not preselected for their EGFR status.The 528 antibody was strongly and homogeneously positive in all samplesexcept two (No. 27 and 29) (44/46, 95.7%). These two cases were alsonegative for mAb806 and mAb DH8.3. The mAb 806 was positive in 27/46(58.7%) cases, 22 of which displayed homogeneous immunoreactivity inmore than 50% of the tumor. The DH8.3 antibody was positive in 15/46(32.6%) glioblastomas, 9 of which showed homogeneous immunoreactivity.The immunochemical staining of these unselected tumors is tabulated inTable 5.

There was concordance between mAb 806 and DH8.3 in every case except one(No. 35).

A molecular analysis for the presence of EGFR amplification was done in44 cases (Table 5). Of these, 30 cases co-typed with the previouslyestablished mAb 806 immunoreactivity pattern: e.g. 16 mAb 806-negativecases revealed no EGFR amplification and 14 EGFR-amplified cases werealso mAb 806 immunopositive. However, 13 cases, which showed 806immunoreactivity, were negative for EGFR amplification while 1EGFR-amplified case was mAb 806 negative. Further analysis of themutation status of these amplification negative and 806 positive casesis described below and provides explanation for most of the 13 caseswhich were negative for EGFR amplification and were recognized by 806.

Subsequently, a molecular analysis of the deletion mutation by RT-PCRwas performed on 41/46 cases (Table 5). Of these, 34 cases co-typed withDH8.3 specific for the deletion mutation: 12 cases were positive in bothRT-PCR and immunohistochemistry and 22 cases were negative/negative.Three cases (#2, #34, #40) were DH8.3 positive/RT-PCR negative for thedeletion mutation and three cases (#12, #18, #39) were DH8.3negative/RT-PCR positive. As expected based on our previous specificityanalysis, mAb 806 immunoreactivity was seen in all DH8.3-positivetissues except in one case (#35).

Case #3 also revealed a mutation (designated A2 in Table 5), whichincluded the sequences of the de2-7 mutation but this did not appear tobe the classical de2-7 deletion with loss of the 801 bases (data notshown). This case was negative for DH8.3 reactivity but showedreactivity with 806, indicating that 806 may recognize an additional andpossibly unique EGFR mutation.

TABLE 5 Immunohistochemical analysis of 46 unselected glioblastomas withmAbs 528, 806, and DH8.3 EGFR # 528 806 DH8.3 Amp.* 5′ MUT 1 ++++ ++++++ A 5′ MUT 2 ++++ ++++ ++++ N WT 3 ++++ ++++ neg. N A2 (det.) 4 ++++++++ neg. N WT 5 ++++ ++++ ++++ N 5′ MUT 6 ++++ ++++ neg. A WT 7 ++++++++ ++++ N 5′ MUT 8 ++++ ++++ ++++ A 5′ MUT 9 ++++ ++++ neg. A WT 10++++ neg. neg. N WT 11 ++ ++ ++ A 5′ MUT 12 ++++ ++ neg. A 5′ MUT 13++++ ++++ neg N WT 14 ++ neg. neg. Nd nd 15 ++ ++ neg N WT 16 + neg.neg. N nd 17 ++++ neg. neg. N WT 18 ++++ ++++ neg. A 5′ MUT 19 ++++ ++++neg. N WT 20 ++++ neg. neg N WT 21 ++++ ++++ neg. N WT 22 +++ neg. neg.N WT 23 ++++ ++++ ++ N 5′ MUT 24 ++++ ++++ neg. A WT 25 ++++ neg. neg. NWT 26 ++++ ++++ +++ A 5′ MUT 27 neg. neg. neg. N WT 28 +++ neg. neg. NWT 29 neg. neg. neg. N WT 30 ++++ ++++ neg. N WT 31 ++++ neg. neg. N ndpar det 32 ++ +++ ++ N 5′ MUT 33 +++ ++++ ++++ A 5′ MUT 34 ++++ +++ ++++N WT 35 ++++ neg. ++++ A 5′ MUT 36 +++ ++ +++ A 5′ MUT 37 ++++ + + A 5′MUT 38 ++++ neg. neg. N WT 39 ++ neg. neg. N 5′ MUT 40 ++++ ++++ + A WT41 ++ neg. neg. N WT 42 ++++ ++++ neg. A WT 43 ++++ neg. neg. nd nd 44++++ neg. neg. N WT 45 ++++ neg. neg. N WT 46 ++++ neg. neg. N nd *N =not amplified, A—amplified, ⁺ WT = wildtype, 5′-mut nd = not done

The 806 antibody reactivity co-typed with amplified or de2-7 mutant EGFRin 19/27 or over 70% of the cases. It is notable that 2 of these 8 caseswere also DH8.3 reactive.

EXAMPLE 16 Systemic Treatment and Analysis of Intracranial Glioma Tumors

To test the efficacy of the anti-ΔEGFR monoclonal antibody, mAb 806, wetreated nude mice bearing intracranial ΔEGFR-overexpressing gliomaxenografts with intraperitoneal injections of mAb806, the isotypecontrol IgG or PBS.

The human glioblastoma cell lines U87MG, LN-Z308 and A1207 (gift fromDr. S. Aaronson, Mount Sinai Medical Center, New York, N.Y.) wereinfected with ΔEGFR, kinase-deficient ΔEGFR (DK), or wild-type EGFR(wtEGFR) viruses. Populations expressing similar high levels of EGFRswere selected by fluorescence-activated cell sorting and designated asU87MG.ΔEGFR, U87MG.DK, U87MG.wtEGFR, LN-Z308.ΔEGFR, LN-Z308.DK, LN-Z308.wtEGFR, A1207.ΔEGFR, A1207.DK and A1207.wtEGFR, respectively. Each wasmaintained in medium containing G418 (U87MG cell lines, 400 μg/ml;LN-Z308 and A1207 cell lines, 800 μg/ml).

U87MG.ΔEGFR cells were implanted intracranially into nude mice and thetreatments began on the same day. 10⁵ cells in 5 μl PBS were implantedinto the right corpus striatum of nude mice brains. Systemic therapywith mAb 806, or the IgG2b isotype control, was accomplished by i.p.injection of 1 mg of mAbs in a volume of 100 μl every other day frompost-implantation day 0 through 14. For direct therapy of intracerebralU87MG.ΔEGFR tumors, 10 μg of mAb 806, or the IgG2b isotype control, in avolume of 5 μl were injected at the tumor-injection site every other daystarting at day 1 for 5 days.

Animals treated with PBS or isotype control IgG had a median survival of13 days, whereas mice treated with mAb 806 had a 61.5% increase inmedian survival up to 21 days (P<0.001).

Treatment of mice 3 days post-implantation, following tumorestablishment, also extended the median survival of the mAb 806 treatedanimals by 46.1% (from 13 days to 19 days; P<0.01) compared to that ofthe control groups.

To determine whether these antitumor effects of mAb 806 extended beyondU87MG.ΔEGFR xenografts, similar treatments were administered to animalsbearing other glioma cell xenografts of LN-Z308.ΔEGFR and A 1207.ΔEGFR.The median survival of mAb 806 treated mice bearing LN-Z308.ΔEGFRxenografts was extended from 19 days for controls to 58 days (P<0.001).Remarkably, four of eight mAb 806 treated animals survived beyond 60days. The median survival of animals bearing A1207.ΔEGFR xenografts wasalso extended from 24 days for controls to 29 days (P<0.01).

MAb 806 Treatment Inhibits ΔEGFR-Overexpressing Brain Tumor Growth.

Mice bearing U87MG.ΔEGFR and LN-Z308.ΔEGFR xenografts were euthanized atday 9 and day 15, respectively. Tumor sections were histopathologicallyanalyzed and tumor volumes were determined Consistent with the resultsobserved for animal survival, mAb 806 treatment significantly reducedthe volumes by about 90% of U87MG.ΔEGFR. (P<0.001) and LN-Z308.ΔEGFR bymore than 95% (P<0.001) xenografts in comparison to that of the controlgroups. Similar results were obtained for animals bearing A1207.ΔEGFRtumors (65% volume reduction, P<0.01).

Intratumoral Treatment with mAb 806 Extends Survival of Mice BearingU87MG.ΔEGFR Brain Tumors.

The efficacy of direct intratumoral injection of mAb 806 for thetreatment of U87MG.ΔEGFR xenografts was also determined Animals weregiven intratumoral injections of mAb 806 or isotype control IgG one daypost-implantation. Control animals survived for 15 days, whereas mAb 806treated mice remained alive for 18 days (P<0.01). While the intratumoraltreatment with mAb 806 was somewhat effective, it entailed thedifficulties of multiple intracranial injections and increased risk ofinfection. We therefore focused on systemic treatments for furtherstudies.

MAb 806 Treatment Slightly Extends Survival of Mice Bearing U87MG.wtEGFRbut not U87MG or U87MG.DK Intracranial Xenografts.

To determine whether the growth inhibition by mAb 806 was selective fortumors expressing ΔEGFR, we treated animals bearing U87MG, U87MG.DK(kinase-deficient ΔEGFR) and U87MG.wtEGFR brain xenografts. MAb 806treatment did not extend survival of mice implanted with U87MG tumorswhich expressed a low level of endogenous wild-type EGFR (wtEGFR), oranimals bearing U87MG.DK xenografts which overexpressed akinase-deficient ΔEGFR in addition to a low level of endogenous wtEGFR.The mAb 806 treatment slightly extended the survival of mice bearingU87MG.wtEGFR tumors (P<0.05,median survival 23 days versus 26 days forthe control groups) which overexpressed wtEGFR

MAb 806 Reactivity Correlates with In Vivo Anti-Tumor Efficacy.

To understand the differential effect of mAb 806 on tumors expressingvarious levels or different types of EGFR, we determined mAb 806reactivity with various tumor cells by FACS analysis. Consistent withprevious reports, the anti-EGFR monoclonal antibody 528 recognized bothΔEGFR and wtEGFR, and demonstrated stronger staining for U87MG.ΔEGFRcells compared to U87MG cells. In contrast, antibody EGFR.1 reacted withwtEGFR but not ΔEGFR, as U87MG.ΔEGFR cells were as weakly reactive asU87MG cells. This EGFR.1 antibody reacted with U87MG.wtEGFR moreintensively than U87MG cells, as U87MG.wtEGFR cells overexpressedwtEGFR. While mAb 806 reacted intensely with U87MG.ΔEGFR and U87MG.DKcells and not with U87MG cells, it reacted weakly with U87MG.wtEGFR,indicating that mAb 806 is selective for ΔEGFR with a weakcross-activity to overexpressed wtEGFR. This level of reactivity withU87MG.wtEGFR was quantitatively and qualitatively similar to theextension of survival mediated by the antibody treatment.

We further determined mAb 806 specificity by immunoprecipitation. EGFRsin various cell lines were immunoprecipitated with antibody 528, EGFR.1and mAb 806. Blots of electrophoretically-separated proteins were thenprobed with the anti-EGFR antibody, C13, which recognizes wtEGFR as wellas ΔEGFR and DK. Consistent with the FACS analysis antibody 528recognized wtEGFR and mutant receptors, while antibody EGFR.1 reactedwith wtEGFR but not the mutant species. Moreover, the levels of mutantreceptors in U87MG.ΔEGFR and U87MG.DK cells are comparable to those ofwtEGFR in the U87MG.wtEGFR cells. However, antibody mAb 806 was able toprecipitate only a small amount of the wtEGFR from the U87MG.wtEGFR celllysates as compared with the larger amount of mutant receptorprecipitated from U87MG.ΔEGFR and U87MG.DK cells, and an undetectableamount from the U87MG cells. Collectively, these data suggest that mAb806 recognizes an epitope in ΔEGFR which also exists in a small fractionof wtEGFR only when it is overexpressed on cell surface.

MAb 806 Treatment Reduces ΔEGFR Autophosphorylation and Down-RegulatesBcl.X_(L) Expression in U87MG.ΔEGFR Brain Tumors.

We next investigated the mechanisms underlying the growth inhibition bymAb 806. Since the constitutively active kinase activity andautophosphorylation of the carboxyl terminus of ΔEGFR are essential forits biological functions we determined ΔEGFR phosphorylation status intumors from treated and control animals. It was found that mAb 806treatment dramatically reduced ΔEGFR autophosphorylation, even thoughreceptor levels were only slightly decreased in the mAb 806 treatedxenografts. We have previously shown that receptor autophosphorylationcauses up-regulation of the antiapoptotic gene, Bcl-X_(L), which plays akey role in reducing apoptosis of ΔEGFR overexpressing tumors.Therefore, we next determined the effect of mAb 806 treatment onBcl-X_(L), expression. ΔEGFR tumors from mAb 806 treated animals didindeed show reduced levels of Bcl-X_(L).

MAb 806 Treatment Decreases Growth and Angiogenesis, and IncreasesApoptosis in U87MG.ΔEGFR Tumors.

In light of the in vivo suppression caused by mAb 806 treatment and itsbiochemical effects on receptor signaling, we determined theproliferation rate of tumors from control or treated mice. Theproliferative index, measured by Ki-67 staining of the mAb 806-treatedtumors, was significantly lower than that of the control tumors(P<0.001). In addition, analysis of the apoptotic index through TUNELstaining demonstrated a significant increase in the number of apoptoticcells in mAb 806 treated tumors as compared with the control tumors(P<0.001). The extent of tumor vascularization was also analyzed byimmunostaining of tumors from treated and control specimens for CD31. Toquantify tumor vascularization, microvascular areas (MVA) were measuredusing computerized image analysis. MAb 806 treated tumors showed 30%less MVA than control tumors (P<0.001). To understand whetherinteraction between receptor and antibody may elicit an inflammatoryresponse, we stained tumor sections for the macrophage marker, F4/80,and the NK cell marker, asialo GM1. Macrophages were identifiedthroughout the tumor matrix and especially accumulated around the mAb806 treated-U87MG.ΔEGFR tumor periphery. We observed a few NK cellsinfiltrated in and around the tumors and no significant differencebetween mAb 806 treated and isotype-control tumors.

EXAMPLE 17 Combination Immunotherapy with mAb806 and mAb528

The experiments set forth herein describe in vivo work designed todetermine the efficacy of antibodies in accordance with this invention.

Female nude mice, 4-6 weeks old, were used as the experimental animals.Mice received subcutaneous inoculations of 3×10⁶ tumor cells in each oftheir flanks. The animals received either U87MG.Δ2-7, U87MG.DK, or A431cells, all of which are described, supra. Therapy began when tumors hadgrown to a sufficient size.

Mice then received injections of one of (i) phosphate buffered saline,(ii) mAb 806 (0.5 mg/injection), (iii) mAb 528 (0.5 mg/injection), or(iv) a combination of both mAbs. With respect to “(iv),” differentgroups of mice received either 0.5 mg/injection of each mAb, or 0.25mg/injection of each mAb.

The first group of mice examined were those which had receivedU87MG.Δ2-7 injections. The treatment protocol began 9 days afterinoculation, and continued, 3 times per week for 2 weeks (i.e., theanimals were inoculated 9, 11, 13, 16, 18 and 20 days after they wereinjected with the cells). At the start of the treatment protocol, theaverage tumor diameter was 115 mm³. Each group contained 50 mice, eachwith two tumors.

Within the group of mice which received the combination of antibodies(0.5 mg/injection of each), there were three complete regressions. Therewere no regressions in any of the other groups. FIG. 18A shows theresults graphically.

In a second group of mice, the injected materials were the same, exceptthe combination therapy contained 0.25 mg of each antibody perinjection. The injections were given 10, 12, 14, 17, 19 and 21 daysafter inoculation with the cells. At the start of the therapy theaverage tumor size was 114 mm³ Results are shown in FIG. 18B.

The third group of mice received inoculations of U87MG.DK. Therapeuticinjections started 18 days after inoculation with the cells, andcontinued on days 20, 22, 25, 27 and 29. The average tumor size at thestart of the treatment was 107 mm³ FIG. 18C summarizes the results. Thetherapeutic injections were the same as in the first group.

Finally, the fourth group of mice, which had been inoculated with A431cells, received injections as in groups I and III, at 8, 10, 12 and 14days after inoculation. At the start, the average tumor size was 71 mm³.Results are shown in FIG. 18D.

The results indicated that the combination antibody therapy showed asynergistic effect in reducing tumors. See FIG. 18A. A similar effectwas seen at a lower dose, as per FIG. 18B, indicating that the effect isnot simply due to dosing levels.

The combination therapy did not inhibit the growth of U87MG.DK (FIG.18C), indicating that antibody immune function was not the cause for thedecrease seen in FIGS. 18A and 18B.

It is noted that, as shown in FIG. 18D, the combination therapy alsoexhibited synergistic efficacy on A431 tumors, with 4 doses leading to a60% complete response rate. These data suggest that the EGFR moleculerecognized by mAb806 is functionally different from that inhibited by528.

References

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EXAMPLE 18 Novel Monoclonal Antibody Specific for the De2-7 EpidermalGrowth Factor Receptor (EGFR) that Also Recognizes the EGFR Expressed inCells Containing Amplification of the EGFR Gene

The following experiments were presented in Johns et al, (2002) Int. J.Cancer, 98, and in co-pending application Ser. No. 60/342,258 filed Dec.21, 2001, the entire disclosure of both of which are incorporated hereinby reference with cross referencing to the Figures herein whereappropriate. The monoclonal antibody mAb 806 was studied and additionaldata respecting its binding characteristics as to the EGF receptor weredeveloped, which is in addition to and corroborative of the datapresented earlier herein. Accordingly, the following represents a reviewand presentation of the material set forth in the patent application andcorresponding publication.

Monoclonal antibody (MAb 806) potentially overcomes the difficultiesassociated with targeting the EGFR expressed on the surface of tumorcells. MAb 806 bound to de2-7 EGFR transfected U87MG glioma cells(U87MG.Δ2-7) with high affinity (˜1×10⁹ M⁻¹), but did not bind parentalcells that express the wild type EGFR. Consistent with this observation,MAb 806 was unable to bind a soluble version of the wild type EGFRcontaining the extracellular domain. In contrast, immobilization of thisextracellular domain to ELISA plates induced saturating and doseresponse binding of MAb 806, suggesting that MAb 806 can bind the wildtype EGFR under certain conditions. MAb 806 also bound to the surface ofA431 cells, which due to an amplification of the EGFR gene express largeamounts of the EGFR. Interestingly, MAb 806 only recognized 10% of thetotal EGFR molecules expressed by A431 cells and the binding affinitywas lower than that determined for the de2-7 EGFR. MAb 806 specificallytargeted U87MG.Δ2-7 and A431 xenografts grown in nude mice with peaklevels in U87MG.Δ2-7 xenografts detected 8 h after injection. Nospecific targeting of parental U87MG xenografts was observed. Followingbinding to U87MG.Δ2-7 cells, MAb 806 was rapidly internalized bymacropinocytosis and subsequently transported to lysosomes, a processthat probably contributes to the early targeting peak observed in thexenografts. Thus, MAb 806 can be used to target tumor cells containingamplification of the EGFR gene or de2-7 EGFR but does not bind to thewild type EGFR when expressed on the cell surface.

As discussed above, MAb 806 is specific for the de2-7 EGFR yet binds toan epitope distinct from the unique junctional peptide. Interestingly,while MAb 806 did not recognize the wild type EGFR expressed on the cellsurface of glioma cells, it did bind to the extracellular domain of thewild type EGFR immobilized on the surface of ELISA plates. Furthermore,MAb 806 bound to the surface of A431 cells, which have an amplificationof the EGFR gene but do not express the de2-7 EGFR. Therefore, it ispossible that MAb 806 could be used to specifically target tumors withamplified EGFR regardless of their de2-7 EGFR status, although ourresults suggest tumors coexpressing the mutated receptor would stillshow preferential targeting. As MAb 806 does not bind wild type receptorin the absence of gene amplification, there would be no uptake in normaltissue, a potential problem associated with EGFR antibodies currentlybeing developed.^(18,19)

Material and Methods

MAbs and Cell Lines

The U87MG astrocytoma cell line has been described in detailpreviously.²⁰ This cell line was infected with a retrovirus containingthe de2-7 EGFR to produce the U87MG.Δ2-7 cell line. 10 Human squamouscarcinoma A431 cells were obtained from ATCC (Rockville, Md.). Thesecell lines were cultured in DMEM/F-12 with GlutaMAX™ (Life Technologies,Melbourne, Australia) supplemented with 10% FCS(CSL, Melbourne,Australia). The murine pro-B cell line BaF/3, which does not express anyknown EGFR related molecules, was transfected with de2-7 EGFR asdescribed above. The DH8.3 antibody (IgG1) has been described previouslyand was obtained following immunization of mice with the uniquejunctional peptide found in de2-7 EGFR.¹⁶ MAb 806 (IgG2b) was producedfollowing immunization of mice with NR6 mouse fibroblasts transfectedwith the de2-7 EGFR. It was selected for further characterization ashemagglutination assays showed a high titer against NR6.ΔEGFR cells butlow backgrounds on NR6.wtEGFR cells. The 528 antibody, which recognizesboth de2-7 and wild type EGFR, has been described previously²¹ and wasproduced in the Biological Production Facility (Ludwig Institute forCancer Research, Melbourne) using a hybridoma obtained from ATCC. Thepolyclonal antibody sc-03 directed to the COOH-terminal domain of theEGFR was purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Other Reagents

The recombinant extracellular domain (amino acids 1-621) of the wildtype EGFR (sEGFR) was produced as previously described.²² Thebiotinylated unique junctional peptide (Biotin-LEEKKGNYVVTDH) (SEQ IDNO:5) from de2-7 EGFR was synthesized by standard Fmoc chemistry andpurity (>96%) determined by reverse phase HPLC and mass spectralanalysis (Auspep, Melbourne, Australia).

FACS Analysis

Cells were labeled with the relevant antibody (10 μg/ml) followed byfluorescein-conjugated goat anti-mouse IgG (1:100 dilution; Calbiochem,San Diego, Calif.). FACS data was obtained on a Coulter Epics ELITE™ ESPby observing a minimum of 5,000 events and analyzed using EXPO™ (version2) for Windows.

ELISA Assays

Two types of ELISA were used to determine the specificity of theantibodies. In the first assay, plates were coated with Segfr (10 μg/mlin 0.1 M carbonate buffer pH 9.2) for 2 hr and then blocked with 2%human serum albumin (HSA) in PBS. Antibodies were added to wells intriplicate at increasing concentration in 2% HSA in phosphate-bufferedsaline (PBS). Bound antibody was detected by horseradish peroxidaseconjugated sheep anti-mouse IgG (Silenus, Melbourne, Australia) usingABTS (Sigma, Sydney, Australia) as a substrate and the absorbancemeasured at 405 nm. In the second assay, the biotinylated de2-7 specificpeptide was bound to ELISA plates precoated with streptavidin (Pierce,Rock-ford, Ill.). Antibodies were bound and detected as in the firstassay.

Scatchard Analysis

Antibodies were labeled with ¹²⁵I (Amrad, Melbourne, Australia) by thechloramine T method and immunoreactivity determined by Lindmo assay. 23All binding assays were performed in 1% HSA/PBS on 1−2×10⁶ liveU87MG.Δ2-7 or A431 cells for 90 min at 4° C. with gentle rotation. A setconcentration of 10 ng/ml ¹²⁵I-labeled antibody was used in the presenceof increasing concentrations of the appropriate unlabeled antibody.Non-specific binding was determined in the presence of 10,000-foldexcess of unlabeled antibody. Neither ¹²⁵I-radiolabeled MAb 806 or theDH8.3 antibody bound to parental U87MG cells. After the incubation wascompleted, cells were washed and counted for bound ¹²⁵I-labeled antibodyusing a COBRA II™ gamma counter (Packard Instrument Company, Meriden,Conn.). Scatchard analysis was done following correction forimmunoreactivity.

Internalization Assay

U87MG.Δ2-7 cells were incubated with either MAb 806 or the DH8.3antibody (10 μg/ml) for 1 hr in DMEM at 4° C. After washing, cells weretransferred to DMEM pre-warmed to 37° C. and aliquots taken at varioustime points following incubation at 37° C. Internalization was stoppedby immediately washing aliquots in ice-cold wash buffer (1% HSA/PBS). Atthe completion of the time course cells were stained by FACS asdescribed above. Percentage internalization was calculated by comparingsurface antibody staining at various time points to zero time using theformula: percent antibody internalized=(mean fluorescence attime_(x)−background fluorescence)/(mean fluorescence at time0_backgroundfluorescence)×100. This method was validated in 1 assay using aniodinated antibody (MAb 806) to measure internalization as previouslydescribed.²⁴ Differences in internalization rate at different timepoints were compared using Student's t-test.

Electron Microscopy of U87MG.Δ2-7 Cells

U87MG.Δ2-7 cells were grown on gelatin coated chamber slides (Nunc,Naperville, Ill.) to 80% confluence and then washed with ice cold DMEM.Cells were then incubated with MAb 806 or the DH8.3 antibody in DMEM for45 min at 4° C. After washing, cells were incubated for a further 30 minwith gold-conjugated (20 nm particles) anti-mouse IgG (BBInternational,Cardiff, UK) at 4° C. Following a further wash, pre-warmed DMEM/10% FCSwas added to the cells, which were incubated at 37° C. for various timesfrom 1-60 min. Internalization of the antibody was stopped by ice-coldmedia and cells fixed with 2.5% glutaraldehyde in PBS/0.1% HSA and thenpostfixed in 2.5% osmium tetroxide. After dehydration through a gradedseries of acetone, samples were embedded in Epon/Araldite resin, cut asultrathin sections with a Reichert Ultracut-S microtome (Leica) andcollected on nickel grids. The sections were stained with uranyl acetateand lead citrate before being viewed on a Philips CM12 transmissionelectron microscope at 80 kV. Statistical analysis of gold grainscontained within coated pits was performed using a χ² test.

Immunoprecipitation Studies

Cells were labeled for 16 hr with 100 μCi/ml of Tran35 S-Label (ICNBiomedicals, CA) in DMEM without methionine/cysteine supplemented with5% dialyzed FCS. After washing with PBS, cells were placed in lysisbuffer (1% Triton X-100, 30 mM HEPES, 150 mM NaCl, 500 μM AEBSF, 150 nMaprotinin, 1 μM E-64 protease inhibitor, 0.5 mM EDTA and 1 μM leupeptin,pH 7.4) for 1 hr at 4° C. Lysates were clarified by centrifugation for10 min at 12,000 g, then incubated with 5 μg of appropriate antibody for30 min at 4° C. before the addition of Protein A-Sepharose™Immunoprecipitates were washed 3 times with lysis buffer, mixed with SDSsample buffer, separated by gel electrophoresis using a 4-20%Tris/glycine gel that was then dried and exposed to X-ray film.

Biodistribution in Tumor Bearing Nude Mice

Tumor xenografts were established in nude BALB/c mice by s.c. injectionof 3×10⁶ U87MG, U87MG.Δ2-7 or A431 cells. de2-7 EGFR expression inU87MG.Δ2-7 xenografts remains stable throughout the period ofbiodistribution as measured by immunohistochemistry at various timepoints (data not shown). A431 cells also retained their MAb 806reactivity when grown as tumor xenografts as determined byimmunohistochemistry. U87MG or A431 cells were injected on 1 side 7-10days before U87MG.Δ2-7 cells were injected on the other side because ofthe faster growth rate observed for de2-7 EGFR expressing xenografts.Antibodies were radiolabeled and assessed for immunoreactivity asdescribed above and were injected into mice by the retro-orbital routewhen tumors were 100-200 mg in weight. Each mouse received 2 differentantibodies (2 μg per antibody): 2 μCi of ¹²⁵I-labeled MAb 806 and 2 μCiof ¹³¹I-labeled DH8.3 or 528. Unless indicated, groups of 5 mice weresacrificed at various time points post-injection and blood obtained bycardiac puncture. The tumors, liver, spleen, kidneys and lungs wereobtained by dissection. All tissues were weighed and assayed for ¹²⁵Iand ¹³¹I activity using a dual-channel counting window. Data wasexpressed for each antibody as percentage injected dose per gram tumor(% ID/g tumor) determined by comparison to injected dose standards orconverted into tumor to blood/liver ratios (i.e., % ID/g tumor÷% ID/gblood or liver). Differences between groups were analyzed by Student'st-test. After injection of radiolabeled MAb 806, some tumors were fixedin formalin, embedded in paraffin, cut into 5 μm sections and thenexposed to X-ray film (AGFA, Mortsel, Belgium) to determine antibodylocalization by autoradiography.

Results

Binding of Antibodies to Cell Lines

In order to confirm the specificity of MAb 806 and the DH8.3 antibody,binding to U87MG and U87MG.Δ2-7 cells was analyzed by FACS. Anirrelevant murine IgG2b was included as an isotype control for MAb 806and the 528 antibody was included as it recognizes both the de2-7 andwild type EGFR. Only the 528 antibody was able to stain the U87MG cellline (FIG. 1) consistent with previous reports demonstrating that thesecells express the wild type EGFR.¹⁰ Both MAb 806 and the DH8.3 antibodyhad binding levels similar to the irrelevant antibody, clearlydemonstrating they are unable to bind the wild type receptor (FIG. 1).Binding of the isotype control antibody to U87MG.Δ2-7 cells was similaras that observed for the U87MG cells. MAb 806 and the DH8.3 antibodyimmunostained U87MG.Δ2-7 cells, indicating that these antibodiesspecifically recognize the de2-7 EGFR (FIG. 1). The 528 antibody stainedU87MG.Δ2-7 with a higher intensity than the parental cell as it bindsboth the de2-7 and wild type receptors that are co-expressed in thesecells (FIG. 1). Importantly, MAb 806 also bound the BaF/3.Δ2-7 cellline, demonstrating that the co-expression of wild type EGFR is not arequirement for MAb 806 reactivity (FIG. 1 but data not shown herein).

Binding of Antibodies in ELISA Assays

To further characterize the specificity of MAb 806 and the DH8.3antibody, their binding was examined by ELISA. Both MAb 806 and the 528antibody displayed dose-dependent and saturating binding curves toimmobilized wild type sEGFR (FIG. 2A). As the unique junctional peptidefound in the de2-7 EGFR is not contained within the sEGFR, MAb 806 mustbe binding to an epitope located within the wild type EGFR sequence. Thebinding of the 528 antibody was probably lower than that observed forMAb 806 as it recognizes a conformational determinant. As expected theDH8.3 antibody did not bind the wild type sEGFR even at concentrationsup to 10 μg/ml (FIG. 2A). Although sEGFR in solution inhibited thebinding of the 528 antibody to immobilized sEGFR in a dose-dependentfashion, it was unable to inhibit the binding of MAb 806 (FIG. 2B). Thissuggests that MAb 806 can only bind wild type EGFR once immobilized onELISA plates, a process that may induce conformational changes. Similarresults were observed using a BIAcore™ whereby MAb 806 bound immobilizedsEGFR but immobilized MAb 806 was not able to bind sEGFR in solution(data not shown). Following denaturation by heating for 10 min at 95°C., sEGFR in solution was able to inhibit the binding of MAb 806 toimmobilized sEGFR (FIG. 2C but data not shown herein), confirming thatMAb 806 can bind the wild type EGFR under certain conditions.Interestingly, the denatured sEGFR was unable to inhibit the binding ofthe 528 antibody (FIG. 2C but data not shown herein, demonstrating thatthis antibody recognizes a conformational epitope. The DH8.3 antibodyexhibited dose-dependent and saturable binding to the unique de2-7 EGFRpeptide (FIG. 2D). Neither MAb 806 or the 528 antibody bound to thepeptide, even at concentrations higher than those used to obtainsaturation binding of DH8.3, further indicating MAb 806 does notrecognize an epitope determinant within this peptide.

Scatchard Analysis of Antibodies

A Scatchard analysis was performed using U87MG.Δ2-7 cells in order todetermine the relative affinity of each antibody. Both MAb 806 and theDH8.3 antibody retained high immunoreactivity when iodinated and wastypically greater than 90% for MAb 806 and 45-50% for the DH8.3antibody. MAb 806 had an affinity for the de2-7 EGFR receptor of 1.1×10⁹M⁻¹ whereas the affinity of DH8.3 was some 10-fold lower at 1.0×10⁸M⁻¹.Neither iodinated antibody bound to U87MG parental cells. MAb 806recognized an average of 2.4×10⁵ binding sites per cell with the DH8.3antibody binding an average of 5.2×10⁵ sites. Thus, there was not onlygood agreement in receptor number between the antibodies, but also witha previous report showing 2.5×10⁵ de2-7 receptors per cell as measuredby a different de2-7 EGFR specific antibody on the same cell line.25

Internalization of Antibodies by U87MG.Δ2-7 Cells

The rate of antibody internalization following binding to a target cellinfluences both its tumor targeting properties and therapeutic options.Consequently, we examined the internalization of MAb 806 and the DH8.3antibody following binding to U87MG.Δ2-7 cells by FACS. Both antibodiesshowed relatively rapid internalization reaching steady-state levels at10 min for MAb 806 and 30 min for DH8.3 (FIG. 3).

Internalization of DH8.3 was significantly higher both in terms of rate(80.5% of DH8.3 internalized at 10 min compared to 36.8% for MAb806,p<0.01) and total amount internalized at 60 min (93.5% vs. 30.4%,p<0.001). MAb 806 showed slightly lower levels of internalization at 30and 60 min compared to 20 min in all 4 assays performed (FIG. 3). Thisresult was also confirmed using an internalization assay based oniodinated MAb 806 (data not shown).

Electron Microscopy Analysis of Antibody Internalization

Given this difference in internalization rates between the antibodies, adetailed analysis of antibody intracellular trafficking was performedusing electron microscopy. Although the DH8.3 anti-body was internalizedpredominantly via coated-pits (FIG. 19A), MAb 806 appeared to beinternalized by macropinocytosis (FIG. 19B). In fact, a detailedanalysis of 32 coated pits formed in cells incubated with MAb 806revealed that none of them contained antibody. In contrast, around 20%of all coated-pits from cells incubated with DH8.3 were positive forantibody, with a number containing multiple gold grains. A statisticalanalysis of the total number of gold grains contained within coated-pitsfound that the difference was highly significant (p<0.01). After 20-30min both antibodies could be seen in structures that morphologicallyresemble lysosomes (FIG. 19C). The presence of cellular debris withinthese structures is also consistent with their lysosome nature.

Biodistribution of Antibodies in Tumor Bearing Nude Mice

The biodistribution of MAb 806 and the DH8.3 antibody was compared innude mice containing U87MG xenografts on 1 side and U87MG.Δ2-7xenografts on the other. A relatively short time period was chosen forthis study as a previous report demonstrated that the DH8.3 antibodyshows peak levels of tumor targeting between 4-24 hr.¹⁶ In terms of %ID/g tumor, MAb 806 reached its peak level in U87MG.Δ2-7 xenografts of18.6% ID/g tumor at 8 hr (FIG. 4A), considerably higher than any othertissue except blood. Although DH 8.3 also showed peak tumor levels at 8hr, the level was a statistically (p<0.001) lower 8.8% ID/g tumorcompared to MAb 806 (FIG. 4B). Levels of both antibodies slowly declinedat 24 and 48 hr. Autoradiography of U87MG.Δ2-7 xenograft tissue sectionscollected 8 hr after injection with ¹²⁵I-labeled MAb 806 alone, clearlyillustrates localization of antibody to viable tumor (FIG. 20). Neitherantibody showed specific targeting of U87MG parental xenografts (FIGS.4A and 4B). With regards to tumor to blood/liver ratios, MAb 806 showedthe highest ratio at 24 hr for both blood (ratio of 1.3) and liver(ratio of 6.1) (FIGS. 5A and 5B). The DH8.3 antibody had its highestratio in blood at 8 hr (ratio of 0.38) and at 24 hr in liver (ratio of1.5) (FIGS. 5A and 5B), both of which are considerably lower than thevalues obtained for MAb 806.

Binding of MAb 806 to Cells Containing Amplified EGFR

To examine if MAb 806 could recognize the EGFR expressed in cellscontaining an amplified receptor gene, its binding to A431 cells wasanalyzed. Low, but highly reproducible, binding of MAb 806 to A431 cellswas observed by FACS analysis (FIG. 6). The DH8.3 antibody did not bindA431 cells, indicating that the binding of MAb 806 was not the result oflow level de2-7 EGFR expression (FIG. 6). As expected, the anti-EGFR 528antibody showed strong staining of A431 cells (FIG. 6). The average of 3such experiments gave a value for affinity of 9.5×10⁷ M⁻¹ with 2.4×10⁵receptors per cell. Thus the affinity for this receptor was some 10-foldlower than the affinity for the de2-7 EGFR. Furthermore, MAb 806 appearsto only recognize a small portion of EGFR found on the surface of A431cells. Using the 528 antibody approximately 2×10⁶ receptors per cellwere measured, which is in agreement with numerous other studies.²⁶ Toensure that these results were not simply restricted to the A431 cellline, MAb 806 reactivity was examined in 2 other cells lines exhibitingamplification of the EGFR gene. Both the HN5 head and neck cell line²⁷and the MDA-468 breast cancer cell line 28 have been reported to containmultiple copies of the EGFR gene. Consistent with these reports, the 528antibody displayed intense staining of both cell lines (FIG. 21). Aswith the A431 cell line, the MAb 806 clearly stained both cell lines butat a lower level than that observed with the 528 antibody (FIG. 21).Thus, MAb 806 binding is not simply restricted to A431 cells but appearsto be a general observation for cells containing amplification of theEGFR gene.

Immunoprecipitations

MAb 806 reactivity was further characterized by immunoprecipitationusing ³⁵S-labeled cells. The sc-03 antibody (a commercial polyclonalantibody specific for the c-terminal domain of the EGFR)immunoprecipitated 3 bands from U87MG.Δ2-7 cells; a doubletcorresponding to the 2 de2-7 EGFR bands observed in these cells and ahigher molecular weight band corresponding to the wt EGFR (FIG. 22). Incontrast, while MAb 806 immunoprecipitated the 2 de2-7 EGFR bands, thewt EGFR was completely absent. The sc-03 antibody immunoprecipitated asingle band corresponding to the wt EGFR from A431 cells (FIG. 22). TheMAb 806 also immunoprecipitated a single band corresponding to the wtEGFR from A431 cells (FIG. 22) but consistent with the FACS andScatchard data, the amount of EGFR immunoprecipitated by MAb 806 wassubstantially less than the total EGFR present on the cell surface.Given that MAb 806 and the sc-03 immunoprecipitated similar amounts ofthe de2-7 EGFR, this result supports the notion that the MAb 806antibody only recognizes a portion of the EGFR in cells overexpressingthe receptor. An irrelevant IgG2b (an isotype control for MAb 806) didnot immunoprecipitate EGFR from either cell line (FIG. 22). Usingidentical conditions, MAb 806 did not immunoprecipitate the EGFR fromthe parental U87MG cells (data not shown).

In Vivo Targeting of A431 Cells by MAb 806

A second biodistribution study was performed with MAb 806 to determineif it could target A431 tumor xenografts. The study was conducted over alonger time course in order to obtain more information regarding thetargeting of U87MG.Δ2-7 xenografts by MAb 806, which were included inall mice as a positive control. In addition, the anti-EGFR 528 antibodywas included as a positive control for the A431 xenografts, since aprevious study demonstrated low but significant targeting of thisantibody to A431 cells grown in nude mice.²¹ During the first 48 hr, MAb806 displayed almost identical targeting properties as those observed inthe initial experiments (FIG. 7A compared with FIG. 4A). In terms of %ID/g tumor, levels of MAb 806 in U87MG.Δ2-7 xenografts slowly declinedafter 24 hr but always remained higher than levels detected in normaltissue. Uptake in the A431 xenografts was comparatively low, however,there was a small increase in % ID/g tumor during the first 24 hr notobserved in normal tissues such as liver, spleen, kidney and lung (FIG.7A). Uptake of the 528 antibody was low in both xenografts whenexpressed as % ID/g tumor (FIG. 7B). Autoradiography of A431 xenografttissue sections collected 24 hr after injection with ¹²⁵I-labeled MAb806 alone, clearly illustrates localization of antibody to viable tumoraround the periphery of the tumor and not central areas of necrosis(FIG. 23). In terms of tumor to blood ratio MAb 806 peaked at 72 hr forU87MG.42-7 xenografts and 100 hr for A431 xenografts (FIGS. 8A AND 8B).Although the tumor:blood ratio for MAb 806 never surpassed 1.0 withrespect to the A431 tumor, it did increase through-out the entire timecourse (FIG. 8B) and was higher than all other tissues examined (datanot shown) indicating low levels of targeting. The tumor to blood ratiofor the 528 antibody showed a similar profile to MAb 806 although higherlevels were noted in the A431 xenografts (FIGS. 8A AND 8B). MAb 806 hada peak tumor to liver ratio in U87MG.Δ2-7 xenografts of 7.6 at 72 hr,clearly demonstrating preferential uptake in these tumors compared tonormal tissue (FIG. 8C). Other tumor to organ ratios for MAb 806 weresimilar to those observed in the liver (data not shown). The peak tumorto liver ratio for MAb 806 in A431 xenografts was 2.0 at 1 od hr, againindicating a slight preferentially uptake in tumor compared with normaltissue (FIG. 8D).

Discussion

The previously described L8A4 monoclonal antibody directed to the uniquejunctional peptide found in the de2-7 EGFR, behaves in a similar fashionto MAb 806.³⁸ Using U87MG cells transfected with the de2-7 EGFR, thisantibody had a similar internalization rate (35% at 1 hr compared to 30%at 1 hr for MAb 806) and displayed comparable in vivo targeting whenusing 3T3 fibroblasts transfected with de2-7 EGFR (peak of 24% ID/gtumor at 24 hr compared to 18% ID/g tumor at 8 hr for MAb 806).²⁵.

Perhaps the most important advantage of MAb 806 compared to current EGFRantibodies, is that MAb 806 can be directly conjugated to cytotoxicagents. This approach is not feasible with current EGFR specificantibodies as they target the liver and cytotoxic conjugation wouldalmost certainly induce severe toxicity. Conjugation of cytotoxic agentssuch as drugs⁴¹ or radioisotopes⁴² to antibodies has the potential toimprove efficacy and reduce the systemic toxicity of these agents. Theability of a conjugated antibody to mediate tumor kill is dependent uponits potential to be internalized. Thus, the rapid internalizationobserved with MAb 806 in U87MG.Δ2-7 cells, suggests MAb 806 is an idealcandidate for this type of approach.

MAb 806 is novel in that it is the first de2-7 EGFR specific antibodydirected to an epitope not associated with the unique junctionalpeptide. It has superior affinity and better tumor targeting propertiesthan DH8.3, a previously described de2-7 EGFR antibody. An importantproperty, however, is its ability to recognize a subset of EGFRmolecules expressed on the surface of tumor cells exhibitingamplification of the EGFR gene. This suggests that MAb 806 may possess aunique clinical property; the ability to target both de2-7 and amplifiedEGFR but not wild type receptors. If proven correct, this antibody wouldnot target organs such as liver and therefore would be more versatilethan current antibodies directed to the EGFR,^(18,19) which cannot beused for the coupling of cytotoxic agents. Finally, MAb 806 may be auseful reagent for analyzing the conformational changes induced by thetruncation found in de2-7 EGFR.

References

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Monoclonal antibody 806    inhibits the growth of tumor xenografts expressing either the de2-7    or amplified epidermal growth factor receptor (EGFR) but not    wild-type EGFR. Cancer Res 2001; 61:5355-61.-   41. Trail P A, Bianchi A B. Monoclonal antibody drug conjugates in    the treatment of cancer. Curr Opin Immunol 1999; 11:584-8.-   42. DeNardo S J, Kroger L A, DeNardo G L. A new era for radiolabeled    antibodies in cancer? Curr Opin Immunol 1999; 11:563-9.

EXAMPLE 19 Growth Suppression of Intracranial Xenografted GlioblastomasOverexpressing Mutant Epidermal Growth Factor Receptors by SystemicAdministration of Monoclonal Antibody (mAb) 806, a Novel MonoclonalAntibody Directed to the Receptor

This example presents the evaluation of mAb 806 on the growth ofintracranial xenografted gliomas in nude mice. The following correspondsto and was presented in Mishima et al, (2001) Cancer Research,61:5349-5354, and the entire publication is incorporated herein byreference with cross referencing to the Figures herein whereappropriate. and made a part hereof. The data and findings of Mishima etal. are set forth below.

Systemic treatment with mAb 806 significantly reduced the volume oftumors and increased the survival of mice bearing xenografts ofU87MG.ΔEGFR, LN-Z308.ΔEGFR, or A1207 gliomas, each of which expresseshigh levels of ΔEGFR. In contrast, mAb 806 treatment was ineffectivewith mice bearing the parental U87MG tumors, which expressed low levelsof endogenous wild-type EGFR, or U87MG.DK tumors, which expressed highlevels of kinase-deficient ΔEGFR. A slight increase of survival of micexenografted with a wild-type EGFR over-expressing U87MG glioma(U87MG.wtEGFR) was effected by mAb 806 concordant with its weakcross-reactivity with such cells. Treatment of U87MG.ΔEGFR tumors inmice with mAb 806 caused decreases in both tumor growth andangiogenesis, as well as increased apoptosis. Mechanistically, in vivomAb 806 treatment resulted in reduced phosphorylation of theconstitutively active ΔEGFR and caused down-regulated expression of theapoptotic protector, BclX_(L). These data provide preclinical evidencethat mAb 806 treatment may be a useful biotherapeutic agent for thoseaggressive gliomas that express ΔEGFR.

The present example demonstrates that systemic treatment with the novelΔEGFR-specific mAb, mAb 806, causes reduced phosphorylation of theconstitutively active ΔEGFR and thereby suppresses growth ofintracranially implanted gliomas overexpressing this mutant receptor innude mice and extends their survival. The inhibition of tumor growth wasmediated by a decrease in proliferation and angiogenesis and increasedapoptosis of the tumor cells. This suppression affected active signalingby ΔEGFR because intracranial xenografts that were derived from cellsoverexpressing kinase-deficient ΔEGFR (DK), which are recognized equallywell by mAb 806, were not significantly suppressed after the sametherapy.

Materials and Methods

Cell Lines.

Because primary explants of human glioblastomas rapidly lose expressionof amplified, rearranged receptors in culture, no existing glioblastomacell lines exhibit such expression. To force maintenance of expressionlevels comparable with those seen in human tumors, U87MG, LN-Z308, andA1207 (gift from Dr. S. Aaronson, Mount Sinai Medical Center, New York,N.Y.) cells were infected with ΔEGFR, kinase-deficient ΔEGFR (DK), orwtEGFR viruses which also conferred resistance to G418 as describedpreviously (21). Populations expressing similar levels of the variousEGFR alleles (these expression levels correspond approximately to anamplification level of 25 gene copies; human glioblastomas typicallyhave amplification levels from 10 to 50 gene copies of the truncatedreceptor) were selected by FACS as described previously (21) anddesignated asU87MG.ΔEGFR, U87MG.DK, U87MG.wtEGFR, LN-Z308.ΔEGFR,LN-Z308.DK, LN-Z308.wtEGFR, A1207.ΔEGFR, A1207.DK, and A1207.wtEGFR,respectively. Each was maintained in medium containing G418 (U87MG celllines, 400 mg/ml; LN-Z308 and A1207 cell lines, 800 mg/ml). mAbs. mAb806 (IgG2b, k), a ΔEGFR specific mAb, was produced after immunization ofmice with NR6 mouse fibroblasts expressing the ΔEGFR. It was selectedfrom several clones because hemagglutination assays showed that it had ahigh reactivity against NR6.ΔEGFR cells, low reactivity for NR6.wtEGFRcells, and none for NR6 cells.

Immunoprecipitation and Western Blot Analysis.

Cells were lysed with lysis buffer containing 50 mM HEPES (pH 7.5), 150mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 0.1% SDS, 0.5% sodiumdeoxycholate, 10 mM sodium PPi, 1 mM phenylmethylsulfonyl fluoride, 2 mMNa3 VO4, 5 μg/m1leupeptin, and 5 mg/ml aprotinin. Antibodies wereincubated with cell lysates at 4° C. for 1 h before the addition ofprotein-A and -G Sepharose™Immuno-precipitates were washed twice withlysis buffer and once with HNTG buffer [50 mM HEPES (pH 7.5), 150 mMNaCl, 0.1% Triton X-100, and 10% glycerol], electrophoresed, andtransferred to nitrocellulose membranes. Blots were probed with theanti-EGFR antibody, C13, and proteins were visualized using the ECL™chemiluminescent detection system (Amersham Pharmacia Biotech.). ThemAbs used for precipitation were mAb 806, anti-EGFR mAb clone 528(Oncogene Research Products, Boston, Mass.), or clone EGFR.1 (OncogeneResearch Products). A mAb, C13, used for detection of both wild-type andΔEGFR on immunoblots was provided by Dr. G. N. Gill (University ofCalifornia, San Diego, Calif.). Antibodies to Bcl-XL (rabbit poly-clonalantibody; Transduction Laboratories, Lexington, Ky.) and phosphotyrosine(4G10, Upstate Biotechnology, Lake Placid, N.Y.) were used for Westernblot analysis as described previously (26).

Flow Cytometry Analysis.

Cells were labeled with the relevant antibody followed byfluorescein-conjugated goat anti-mouse IgG (1:100 dilution;Becton-Dickinson PharMingen, San Diego, Calif.) as described previously(21). Stained cells were analyzed with a FACSCalibur™ using Cell Quest™software (Becton-Dickinson PharMingen). For the first antibody, thefollowing mAbs were used: mAb 806, anti-EGFR mAb clone 528, and cloneEGFR.1. Mouse IgG2a or IgG2b was used as an isotype control.

Tumor Therapy.

U87MG.ΔEGFR cells 1×10⁵) or 5×10⁵ LN-Z308.ΔEGFR, A1207.ΔEGFR, U87MG,U87MG.DK, and U87MG.wtEGFR cells in 5 μl of PBS were implanted into theright corpus striatum of nude mice brains as described previously (27).Systemic therapy with mAb 806, or the IgG2b isotype control, wasaccomplished by i.p. injection of 1 μg of mAbs in a volume of 100 μlevery other day from postimplantation day 0 through 14. For directtherapy of intracerebral U87MG.ΔEGFR tumors, 10 μg of mAb 806, or theIgG2b isotype control, in a volume of 5 μl were injected at thetumor-injection site every other day starting at day 1 for 5 days.

Immunohistochemistry.

To assess angiogenesis in tumors, they were fixed in a solutioncontaining zinc chloride, paraffin embedded, sectioned, andimmunostained using a monoclonal rat anti-mouse CD31 antibody(Becton-Dickinson PharMingen; 1:200). Assessment of tumor cellproliferation was performed by Ki-67 immunohistochemistry onformalin-fixed paraffin-embedded tumor tissues. After deparaffinizationand rehydration, the tissue sections were incubated with 3% hydrogenperoxide in methanol to quench endogenous peroxidase. The sections wereblocked for 30 min with goat serum and incubated overnight with theprimary antibody at 4° C. The sections were then washed with PBS andincubated with a biotinylated secondary antibody for 30 min. Afterseveral washes with PBS, products were visualized using streptavidinhorseradish peroxidase with diaminobenzidine as chromogen andhematoxylin as the counterstain. As a measure of proliferation, theKi-67 labeling index was determined as the ratio of labeled:total nucleiin high-power (3400) fields. Approximately 2000 nuclei were counted ineach case by systematic random sampling. For macrophage and NK cellstaining, frozen sections, fixed with buffered 4% paraformaldehydesolution, were immunostained using biotinylated mAb F4/80 (Serotec,Raleigh, N.C.) and polyclonal rabbit anti-asialo GM1 antibody (DakoChemicals, Richmond, Va.), respectively. Angiogenesis was quantitated asvessel area using computerized analysis. For this purpose, sections wereimmunostained using anti-CD31 and were analyzed using a computerizedimage analysis system without counterstain. MVAs were determined bycapturing digital images of the sections at 3200 magnification using aCCD color camera as described previously (27). Images were then analyzedusing Image Pro® Plus version 4.0 software (Media Cybernetics, SilverSpring, Md.) and MVA was determined by measuring the total amount ofstaining in each section. Four fields were evaluated for each slide.This value was represented as a percentage of the total area in eachfield. Results were confirmed in each experiment by at least twoobservers (K. M., H-J. S. H.).

TUNEL Assay.

Apoptotic cells in tumor tissue were detected by using the TUNEL methodas described previously (27). TUNEL-positive cells were counted at ×400.The apoptotic index was calculated as a ratio of apoptotic cellnumber:total cell number in each field.

Statistical Analysis.

The data were analyzed for significance by Student's t test, except forthe in vivo survival assays, which were analyzed by Wilcoxon analysis.

Results

Systemic Treatment of mAb 806 Extends the Survival of Mice BearingΔEGFR-Overexpressing Intracranial Glioma Tumors.

To test the efficacy of the anti-ΔEGFR mAb, mAb 806, we treated nudemice bearing intracranial ΔEGFR-overexpressing glioma xenografts withi.p. injections of mAb 806, the isotype control IgG, or PBS. U87MG.ΔEGFRcells were implanted intracranially into nude mice, and the treatmentsbegan on the same day as described in “Materials and Methods.”

Animals treated with PBS or isotype control IgG had a median survival of13 days, whereas mice treated with mAb 806 had a 61.5% increase inmedian survival up to 21 days (P<0.001; FIG. 24A). Treatment of mice 3days postimplantation, after tumor establishment, also extended themedian survival of the mAb 806-treated animals by 46.1% (from 13 days to19 days; P<0.01) compared with that of the control groups (data notshown). To determine whether these antitumor effects of mAb 806 extendedbeyond U87MG.ΔEGFR xenografts, we also did similar treatments of animalsbearing other glioma cell xenografts of LN-Z308.ΔEGFR and A1207.ΔEGFR.The median survival of mAb 806-treated mice bearing LN-Z308.ΔEGFRxenografts was extended from 19 days for controls to 58 days (P<0.001;FIG. 24B). Remarkably, four of eight mAb 806-treated animals survivedbeyond 60 days (FIG. 24B). The median survival of animals bearingA1207.ΔEGFR xenografts was also extended from 24 days for controls to 29days (P<0.01; data not shown).

mAb 806 Treatment Inhibits ΔEGFR-Overexpressing Brain Tumor Growth.

Mice bearing U87MG.ΔEGFR and LN-Z308.ΔEGFR xenografts were killed at day9 and day 15, respectively. Tumor sections were histopathologicallyanalyzed, and tumor volumes were determined as described in “Materialsand Methods.” Consistent with the results observed for animal survival,mAb 806 treatment significantly reduced the volumes of U87MG.ΔEGFR by90% (P<0.001; FIG. 24C), and of LN-Z308.ΔEGFR by 0.95% (P<0.001; FIG.24D), of xenografts in comparison with those of the control groups.Similar results were obtained for animals bearing A1207.ΔEGFR tumors(65% volume reduction; P<0.01; data not shown).

Intratumoral Treatment with mAb 806 Extends Survival of Mice Bearing U87MG.ΔEGFR Brain Tumors.

We also determined the efficacy of direct intratumoral injection of mAb806 for the treatment of U87MG.ΔEGFR xenografts. Animals were givenintratumoral injections of mAb 806 or isotype control IgG at 1 day postimplantation, as described in “Materials and Methods.” Control animalssurvived for 15 days, whereas mAb 806 treated mice remained alive for 18days (P<0.01; FIG. 24E). Although the intratumoral treatment with mAb806 was somewhat effective, it entailed the difficulties of multipleintracranial injections and of increased risk of infection. We,therefore, focused on systemic treatments for additional studies.

mAb 806 Treatment Slightly Extends Survival of Mice Bearing U87MG.wtEGFR but not of Mice Bearing U87 MG or U87 MG.DK IntracranialXenografts.

To determine whether the growth inhibition by mAb 806 was selective fortumors expressing ΔEGFR, we treated animals bearing U87MG, U87MG.DK(kinase-deficient ΔEGFR) or U87MG.wtEGFR brain xenografts. mAb 806treatment did not extend the survival of mice implanted with U87 mgtumors (FIG. 25A), which expressed a low level of endogenous wtEGFR(22), or of animals bearing U87MG.DK xenografts, which overexpressed akinase-deficient ΔEGFR in addition to a low level of endogenous wtEGFR(FIG. 25B). The mAb 806 treatment slightly extended the survival of micebearing U87MG.wtEGFR tumors (P<0.05; median survival, 23 days versus 26days for the control groups), which overexpressed wtEGFR (FIG. 25C).

mAb 806 Reactivity Correlates with in Vivo Antitumor Efficacy.

To understand the differential effect of mAb 806 on tumors expressingvarious levels or different types of EGFR, we determined mAb 806reactivity with various tumor cells by FACS analysis. Consistent withprevious reports (21), the anti-EGFR mAb 528 recognized both ΔEGFR andwtEGFR and demonstrated stronger staining for U87MG.ΔEGFR cells comparedwith U87MG cells (FIG. 26A, 528). In contrast, antibody EGFR.1 reactedwith wtEGFR but not with ΔEGFR (21), because U87MG.ΔEGFR cells were asweakly reactive as U87MG cells (FIG. 26A, panel EGFR.1). This EGFR.1antibody reacted with U87MG.wt.EGFR more intensively than with U87MGcells, because U87MG.wt.EGFR cells overexpressed wtEGFR (FIG. 26A, panelEGFR.1). Although mAb 806 reacted intensely with U87MG.ΔEGFR andU87MG.DK cells and not with U87MG cells, it reacted weakly U87MG.wtEGFR,which indicated that mAb 806 is selective for ΔEGFR with a weakcross-activity to overexpressed wtEGFR (FIG. 26A, panel mAb 806). Thislevel of reactivity with U87MG.wtEGFR was quantitatively andqualitatively similar to the extension of survival mediated by theantibody treatment (FIG. 25C).

We further determined mAb 806 specificity by immunoprecipitation. EGFRsin various cell lines were immunoprecipitated with antibody 528, EGFR.1,and mAb 806. Blots of electrophoretically separated proteins were thenprobed with the anti-EGFR antibody, C13, which recognizes wtEGFR as wellas ΔEGFR and DK (22). Consistent with the FACS analysis, antibody 528recognized wtEGFR and mutant receptors (FIG. 26B-panel IP: 528), whereasantibody EGFR.1 reacted with wtEGFR but not with the mutant species(FIG. 26B, panel IP: EGFR.1). Moreover, the levels of mutant receptorsin U87MG.ΔEGFR and U87MG.DK cells are comparable with those of wtEGFR inthe U87MG.wtEGFR cells (FIG. 26B, panel IP: 528).

However, antibody mAb 806 was able to precipitate only a small amount ofthe wtEGFR from the U87MG.wtEGFR cell lysates as compared with thelarger amount of mutant receptor precipitated from U87MG.ΔEGFR andU87MG.DK cells, and an undetectable amount from the U87MG cells (FIG.26B, panel IP: mAb 806). Collectively, these data suggest that mAb 806recognizes an epitope in ΔEGFR that also exists in a small fraction ofwtEGFR only when it is overexpressed on the cell surface.

mAb 806 Treatment Reduces ΔEGFR Autophosphorylation and Down-RegulatesBcl-XL Expression in U87MG.ΔEGFR Brain Tumors.

The mechanisms underlying the growth inhibition by mAb 806 were nextinvestigated. Because the constitutively active kinase activity andautophosphorylation of the COOH terminus of ΔEGFR are essential for itsbiological functions (21, 22, 28, 29), ΔEGFR phosphorylation status wasdetermined in tumors from treated and control animals. As shown in FIG.27A, mAb 806 treatment dramatically reduced ΔEGFR autophosphorylation,although receptor levels were only slightly decreased in the mAb806-treated xenografts. We have previously shown that receptorautophosphorylation causes up-regulation of the antiapoptotic gene,Bcl-X_(L), which plays a key role in reducing apoptosis ofΔEGFR-overexpressing tumors (28, 29). Therefore, the effect of mAb 806treatment on Bcl-X_(L) expression was next determined ΔEGFR tumors frommAb 806-treated animals did indeed show reduced levels of Bcl-X_(L)(FIG. 27A).

mAb 806 Treatment Decreases Growth and Angiogenesis and IncreasesApoptosis in U87 MG.ΔEGFR Tumors.

In light of the in vivo suppression caused by mAb 806 treatment and itsbiochemical effects on receptor signaling, we determined theproliferation rate of tumors from control or treated mice. Theproliferative index, measured by Ki-67 staining of the mAb 806-treatedtumors, was significantly lower than that of the control tumors(P<0.001; FIG. 28). In addition, analysis of the apoptotic index throughTUNEL staining demonstrated a significant increase in the number ofapoptotic cells in mAb 806-treated tumors as compared with the controltumors (P<0.001; FIG. 28). The extent of tumor vascularization was alsoanalyzed by immunostaining of tumors from treated and control specimensfor CD31. To quantify tumor vascularization, MVAs were measured usingcomputerized image analysis. mAb 806-treated tumors showed 30% less MVAthan did control tumors (P<0.001; FIG. 28). To understand whetherinteraction between receptor and antibody may elicit an inflammatoryresponse, we stained tumor sections for the macrophage marker, F4/80,and the NK cell marker, asialo GM1. Macrophages were identifiedthroughout the tumor matrix and especially accumulated around the mAb806-TREATED-U87MG.ΔEGFR-tumor periphery (FIG. 28). We observed few NKcells infiltrated in and around the tumors and no significant differencebetween mAb 806-treated and isotype-control tumors (data not shown).

Discussion

ΔEGFR appears to be an attractive potential therapeutic target forcancer treatment of gliomas. It is correlated with poor prognosis (25),whereas its genetic or pharmacological inhibition effectively suppressesgrowth of ΔEGFR-overexpressing cells both in vitro and in vivo (29, 30).Because this mutant EGFR is expressed on the cell surface, it representsa potential target for antibody-based therapy, and, here, we tested theefficacy of a novel anti-ΔEGFR mAb, mAb 806, on the treatment ofintracranial xenografts of ΔEGFR-overexpressing gliomas of differentcellular backgrounds in nude mice. The systemic administration of mAb806 inhibited tumor growth and extended animal survival. The effect ofmAb 806 was evident for each cell line and was independent of the p53status of the tumors, because U87MG.ΔEGFR and A1207.ΔEGFR expressedwild-type p53, whereas LN-Z308.ΔEGFR was p53-null.

The enhanced tumorigenicity of ΔEGFR is mediated through itsconstitutively active kinase activity and tyrosine autophosphorylationat the COOH terminus (22, 28, 29). Phosphorylation of ΔEGFR in mAb806-treated tumors was significantly decreased, proliferation wasreduced, and apoptosis was elevated, which suggests that the antitumoreffect of mAb 806 is, at least in part, attributable to the inhibitionof the intrinsic function of the receptor. The ΔEGFR signaling causedup-regulation of the antiapoptotic gene, Bcl-X_(L) (28), and treatmentwith mAb 806 resulted in down-regulation of Bcl-X_(L) expression, whichfurther suggests that the antitumor effect of mAb 806 is mediatedthrough the inhibition of ΔEGFR signaling. The level of ΔEGFR in the mAb806-treated tumors was also slightly reduced (FIG. 27A), but not to adegree that was consistent with the degree of dephosphorylation of themutant receptor or sufficient to explain the magnitude of its biologicaleffect. The antitumor effect of mAb 806 is likely to result, at least inpart, from the inhibition of the intrinsic signaling function of ΔEGFR.This assertion is also supported by the lack of antitumor effects on DKtumors, which bind to the antibody but are kinase deficient.

Intratumoral injection of a different anti-ΔEGFR antibody, mAbY10,inhibited the growth of ΔEGFR-expressing B16 melanoma tumors in mousebrains through a Fc/Fc receptor-dependent mechanism (31). In conjunctionwith this, mAbY10 was shown to mediate antibody-dependent macrophagecytotoxicity in vitro with both murine and human effector cells (17),although it had little effect with macro-phage infiltration found in ourmAb 806-treated tumors raises the question as to whether the antitumoreffect of mAb 806 may be accomplished by macrophage-mediatedcytotoxicity. We believe this to be unlikely, because macrophageinfiltration also occurred on mAb 806 treatment of U87MG.DK(kinase-deficient ΔEGFR) tumors, in which it was ineffective inregulating tumor growth.

mAb 806 appears to be selective for ΔEGFR with a weak cross-reactivitywith overexpressed wtEGFR. Consistent with the in vitro specificity, mAb806 treatment was very effective in ΔEGFR-over-expressed tumors, whereasit showed a much less robust, but reproducible, growth inhibition fortumors overexpressing wtEGFR. However, the simple interaction betweenmAb 806 and its target molecules is insufficient to inhibit tumor growthbecause, although mAb 806 is capable of binding equally well tokinase-deficient ΔEGFR (DK) receptors and ΔEGFR, it is ineffective inaffecting DK-expressing tumor growth. The inability of mAb 806 tointeract with the low-level of wtEGFR normally present in cells suggestsa large therapeutic window for ΔEGFR-overexpressed as well as, to alesser extent, wtEGFR-overexpressed cancers when compared with normaltissues.

Although the mAb 806 treatment was effective for suppression ofintracranial xenografts, it should be noted that the ΔEGFR-tumorseventually grew, and durable remissions were not achieved. This may haveresulted from inefficient distribution of antibody in the tumor mass.mAbs in combination with other therapeutic modalities such as toxins,isotopes or drugs, for cancer treatments have been shown to be moreeffective than antibody alone in many cases (2, 3, 32-34).Chemotherapeutic drugs such as doxorubicin and cisplatin in conjunctionwith wtEGFR antibodies have also shown enhanced antitumor activity (35,36). Combination treatments targeted at tumor growth as well asangiogenic development have more effectively inhibited glioblastomagrowth than either treatment alone (27). This raises the possibilitythat mAb 806 in combination with chemotherapeutic drugs or compoundsmodulating angiogenesis may be even more effective than mAb 806 alone.

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Neurosurgery (Baltimore),    45: 1442-1453, 1999.-   26. Nagane, M., Levitzki, A., Gazit, A., Cavenee, W. K., and Huang,    H-J. S. Drug resistance of human glioblastoma cells conferred by a    tumor-specific mutant epidermal growth factor receptor through    modulation of Bcl-X_(L) and caspase-3-like proteases. Proc. Natl.    Acad. Sci. USA, 95: 5724-5729, 1998.-   27. Mishima, K., Mazar, A. P., Gown, A., Skelly, M., Ji, X. D.,    Wang, X. D., Jones, T. R., Cavenee, W. K., and Huang, H-J. S. A    peptide derived from the non-receptor-binding region of urokinase    plasminogen activator inhibits glioblastoma growth and angiogenesis    in vivo in combination with cisplatin. Proc. Natl. Acad. Sci. USA,    97: 8484-8489, 2000.-   28. Nagane, M., Coufal, F., Lin, H., Bogler, O., Cavenee, W. K., and    Huang, H-J. S. A common mutant epidermal growth factor receptor    confers enhanced tumorigenicity on human glioblastoma cells by    increasing proliferation and reducing apoptosis. Cancer Res., 56:    5079-5086, 1996.-   29. Nagane, M., Lin, H., Cavenee, W. K., and Huang, H-J. S. Aberrant    receptor signaling in human malignant gliomas: mechanisms and    therapeutic implications. Cancer Lett., 162 (Suppl. 1): S17-S21,    2001.-   30. Halatsch, M. E., Schmidt, U., Botefur, I. C., Holland, J. F.,    and Ohnuma, T. Marked inhibition of glioblastoma target cell    tumorigenicity in vitro by retrovirus-mediated transfer of a hairpin    ribozyme against deletion-mutant epidermal growth factor receptor    messenger RNA. J. Neurosurg., 92: 297-305, 2000.-   31. Sampson, J. H, Crotty, L. E., Lee, S., Archer, G. E., Ashley, D.    M., Wikstrand, C. J., Hale, L. P., Small, C., Dranoff, G.,    Friedman, A. H., Friedman, H. S., and Bigner, D. D. Unarmed,    tumor-specific monoclonal antibody effectively treats brain tumors.    Proc. Natl. Acad. Sci. USA, 97: 7503-7508, 2000.-   32. Trail, P. A, and Bianchi, A. B. Monoclonal antibody drug    conjugates in the treatment of cancer. Curr. Opin. Immunol., 11:    584-588, 1999.-   33. Pietras, R. J., Pegram, M. D., Finn, R. S., Maneval, D. A., and    Slamon, D. J. Remission of human breast cancer xenografts on therapy    with humanized monoclonal antibody to HER-2 receptor and    DNA-reactive drugs. Oncogene, 17: 2235-2249, 1998.-   34. Baselga, J., Norton, L., Albanell, J., Kim, Y. M., and    Mendelsohn, J. Recombinant humanized anti-HER2 antibody (Herceptin)    enhances the antitumor activity of pacli-taxel and doxorubicin    against HER2/neu overexpressing human breast cancer xenografts.    Cancer Res., 58: 2825-2831, 1998.-   35. Baselga, J., Norton, L., Masui, H., Pandiella, A., Coplan, K.,    Miller, W. H., and Mendelsohn, J. Antitumor effects of doxorubicin    in combination with anti-epidermal growth factor receptor monoclonal    antibodies. J. Natl. Cancer Inst. (Bethesda), 85: 1327-1333, 1993.-   36. Fan, Baselga, J., Masui, H., and Mendelsohn, J. Antitumor effect    of anti-epidermal growth factor receptor monoclonal antibodies plus    cis-diamminedichloroplatinum on well established A431 cell    xenografts. Cancer Res., 53: 4637-4642, 1993.

EXAMPLE 20 Monoclonal Antibody 806 Inhibits the Growth of TumorXenografts Expressing Either the De2-7 or Amplified Epidermal GrowthFactor Receptor (EGFR) but not Wild-Type EGFR

The following example presents findings by the present inventors that isalso set forth in Luwor et al., (2001) Cancer Research, 61:5355-5361.The disclosure of this publication is incorporated herein in itsentirety with cross referencing to the Figures herein where appropriate.and made a part hereof.

The monoclonal antibody (mAb) 806 was raised against the delta2-7epidermal growth factor receptor (de2-7 EGFR or EGFRvIII), a truncatedversion of the EGFR commonly expressed in glioma. Unexpectedly, mAb 806also bound the EGFR expressed by cells exhibiting amplification of theEGFR gene but not to cells or normal tissue expressing the wild-typereceptor in the absence of gene amplification. The unique specificity ofmAb 806 offers an advantage over current EGFR antibodies, which alldisplay significant binding to the liver and skin in humans. Therefore,we examined the antitumor activity of mAb 806 against human tumorxenografts grown in nude mice. The growth of U87MG xenografts, a gliomacell line that endogenously expresses ˜10⁵ EGFRs in the absence of geneamplification, was not inhibited by mAb 806. In contrast, mAb 806significantly inhibited the growth of U87MG xenografts transfected withthe de2-7 EGFR in a dose-dependent manner using both preventative andestablished tumor models. Significantly, U87MG cells transfected withthe wild-type EGFR, which increased expression to ˜10⁶ EGFRs/cell andmimics the situation of gene amplification, were also inhibited by mAb806 when grown as xenografts in nude mice. Xenografts treated with mAb806 all displayed large areas of necrosis that were absent in controltumors. This reduced xenograft viability was not mediated by receptordown-regulation or clonal selection because levels of antigen expressionwere similar in control and treated groups. The antitumor effect of mAb806 was not restricted to U87MG cells because the antibody inhibited thegrowth of new and established A431 xenografts, a cell line expressing>10⁶ EGFRs/cell. This study demonstrates that mAb 806 possessessignificant antitumor activity.

The de2-7 EGFR specific mAb 806 was produced after immunization of micewith NR6 mouse fibroblasts expressing the truncated de2-7 EGFR. mAb 806binds the U87MG glioma cell line transfected with the de2-7 EGFR but notthe parental U87MG cell line, which expresses the wt EGFR without geneamplification.³ Similar results were observed in vivo with mAb 806showing specific targeting of de2-7 EGFR expressing U87MG xenografts butnot parental U87MG tumors.³ Interestingly, mAb 806 was capable ofbinding an EGFR subset (−10%) on the surface of the A431 cell line,which contains an amplified EGFR gene. Therefore, unlike all other de2-7EGFR-specific antibodies, which recognize the unique peptide junctionthat is generated by the de2-7 EGFR truncation, mAb 806 binds to anepitope also found in overexpressed wt EGFR. However, it would appearthat this epitope is preferentially exposed in the de2-7 EGFR and asmall proportion of receptors expressed in cells containing wt EGFR geneamplification. Importantly, normal tissues that expresses high levels ofendogenous wt EFGR, such as liver and skin, show no significant mAb 806binding. On the basis of the unique property of the mAb 806 to bind boththe de2-7 and amplified wt EGFR but not the native wt EGFR whenexpressed at normal levels, we decided to examine the efficacy of mAb806 against several tumor cell lines grown as xenografts in nude mice.

Materials and Methods

Cell Lines and Monoclonal Antibodies.

The human glioblastoma cell line U87MG, which endogenously expresses thewt EGFR, and the transfected cell lines U87MG.Δ2-7 and U87MG.wtEGFR,which express the de2-7 EGFR and overexpress the wt EGFR, respectively,have been described previously (16, 23). The epidermoid carcinoma cellline A431 has been described previously (24).

All cell lines were maintained in DMEM (DMEM/F12; Life Technologies,Inc., Grand Island, N.Y.) containing 10% FCS (CSL, Melbourne, Victoria,Australia), 2 mM glutamine (Sigma Chemical Co., St. Louis, Mo.), andpenicillin/streptomycin (Life Technologies, Inc., Grand Island, N.Y.).In addition, the U87MG.Δ2-7 and U87MG.wtEGFR cell lines were maintainedin 400 mg/ml of GENETICIN™ (Life Technologies, Inc., Melbourne,Victoria, Australia). Cell lines were grown at 37° C. in a humidifieratmosphere of 5% CO2. The mAb 806 (IgG2b) was produced afterimmunization of mice with NR6 mouse fibroblasts expressing the de2-7EGFR. mAb 806 was selected after rosette assays showed binding to NR6cells, which overexpressed the de2-7 EGFR (titer of 1:2500). mAb 528,which recognizes both de2-7 and wt EGFR, has been described previously(10) and was produced in the Biological Production Facility (LudwigInstitute for Cancer Research, Melbourne, Victoria, Australia) using ahybridoma obtained from American Type Culture Collection (Rockville,Md.). The DH8.3 mAb, which is specific for the de2-7 EGFR, was kindlyprovided by Prof. William Gullick (University of Kent and Canterbury,Kent, United Kingdom) (19). The polyclonal antibody sc-03 directed tothe COOH-terminal domain of the EGFR was purchased from Santa CruzBio-technology (Santa Cruz Biotechnology, Santa Cruz, Calif.).

FACS Analysis of Receptor Expression.

Cultured parental and trans-fected U87MG cell lines were analyzed for wtand de2-7 EGFR expression using the 528, 806, and DH8.3 antibodies.Cells (1 3 10 6) were incubated with 5 mg/ml of the appropriate antibodyor an isotype-matched negative control in PBS containing 1% HSA for 30min at 4° C. After three washes with PBS/1% HSA, cells were incubated anadditional 30 min at 4° C. with FITC-coupled goat antimouse antibody(1:100 dilution; Calbiochem, San Diego, Calif.). After three subsequentwashes, cells were analyzed on an Epics Elite™ ESP (Beckman Coulter,Hialeah, Fla.) by observing a minimum of 20,000 events and analyzedusing EXPO™ (version 2) for Windows.

Scatchard Analysis.

The mAb 806 was labeled with ¹²⁵I (Amrad, Melbourne, Victoria,Australia) by the Chloramine T method. All binding assays were performedin 1% HSA/PBS on 1−2×10⁶ live U87MG.Δ2-7 or A431 cells for 90 min at 4°C. with gentle rotation. A set concentration of 10 ng/ml ¹²⁵I-labeledmAb 806 was used in the presence of increasing concentrations ofunlabeled antibody. Nonspecific binding was determined in the presenceof 10,000-fold excess of unlabeled antibody. After incubation, cellswere washed and counted for bound ¹²⁵I-labeled mAb 806 using a COBRA IIgamma counter (Packard Instrument Company, Meriden, Conn.). Scatchardanalysis was done after correction for immunoreactivity.

Immunoprecipitation Studies.

Cells were labeled for 16 h with 100 mCi/ml of Tran 35 S-Label (ICNBiomedicals, Irvine, Calif.) in DMEM without methionine/cysteinesupplemented with 5% dialyzed FCS. After washing with PBS, cells wereplaced in lysis buffer (1% Triton X-100, 30 mM HEPES, 150 mM NaCl, 500mM 4-(2-aminoethyl)benzenesulfonylfluoride, 150 nM aprotinin, 1 mM E-64protease inhibitor, 0.5 mM EDTA, and 1 mM leupeptin, pH 7.4) for 1 h at4° C. Lysates were clarified by centrifugation for 10 min at 12,000 3 gand then incubated with 5 mg of appropriate antibody for 30 min at 4° C.before the addition of protein A-Sepharose™ Immunoprecipitates werewashed three times with lysis buffer, mixed with SDS sample buffer,separated by gel electrophoresis using a 7.5% gel that was then dried,and exposed to X-ray film.

Xenograft Models.

Consistent with previous reports (23, 25), U87MG cells transfected withde2-7 EGFR grew more rapidly then parental cells and U87MG cellstransfected with the wt EGFR. Tumor cells (3×10⁶) in 100 ml of PBS wereinoculated s.c. into both flanks of 4-6-week-old, female nude mice(Animal Research Center, Western Australia, Perth, Australia).Therapeutic efficacy of mAb 806 was investigated in both preventativeand established tumor models. In the preventative model, five mice withtwo xenografts each were treated i.p. with either 0.1 or 1 mg of mAb 806or vehicle (PBS) starting the day before tumor cell inoculation.Treatment was continued for a total of six doses, three times per weekfor 2 weeks. In the established model, treatment was started when tumorshad reached a mean volume of 65 mm 3 (U87MG.Δ2-7), 84 mm 3 (U87MG), 73mm 3 (U87MG.wtEGFR), or 201 mm 3 (A431 tumors). Tumor volume in mm 3 wasdetermined using the formula (length 3 width 2)/2, where length was thelongest axis and width the measurement at right angles to the length(26). Data were expressed as mean tumor volume 6 SE for each treatmentgroup. This research project was approved by the Animal Ethics Committeeof the Austin and Repatriation Medical Centre.

Histological Examination of Tumor Xenografts.

Xenografts were excised at the times indicated and bisected. One halfwas fixed in 10% formalin/PBS before being embedded in paraffin. Four-mmsections were then cut and stained with H&E for routine histologicalexamination. The other half was embedded in Tissue Tek® O.C.T.™ compound(Sakura Finetek, Torrance, Calif.), frozen in liquid nitrogen, andstored at 280° C. Thin (5-mm) cryostat sections were cut and fixed inice-cold acetone for 10 min, followed by air drying for an additional 10min. Sections were blocked in protein blocking reagent (Lipshaw Immunon,Pittsburgh, Pa.) for 10 min and then incubated with biotinylated primaryantibody (1 mg/ml) for 30 min at room temperature. All antibodies werebiotinylated using the ECL™ protein biotinylation module (Amersham,Baulkham Hills, NSW, Australia), as per the manufacturer's instructions.After rinsing with PBS, sections were incubated with astreptavidin-horseradish peroxidase complex for an additional 30 min(Silenus, Melbourne, Victoria, Australia). After a final PBS wash, thesections were exposed to 3-amino-9-ethylcarbazole substrate [0.1 Macetic acid, 0.1 M sodium acetate, 0.02 M 3-amino-9-ethylcarbazole(Sigma Chemical Co., St. Louis, Mo.)] in the presence of hydrogenperoxide for 30 min. Sections were rinsed with water and counterstainedwith hematoxylin for 5 min and mounted.

Statistical Analysis.

The in vivo tumor measurements in mm 3 are ex-pressed as the mean 6 SE.Differences between treatment groups at given time points were testedfor statistical significance using Student's t test.

Results

Binding of Antibodies to Cell Lines.

To determine the specificity of mAb 806, its binding to U87MG,U87MG.Δ2-7, and U87MG.wtEGFR cells was analyzed by FACS. An irrelevantIgG2b (mAb 100-310 directed to the human antigen A33) was included as anisotype control for mAb 806, and the 528 antibody was included becauseit recognizes both the de2-7 and wt EGFR. Only the 528 antibody was ableto stain the parental U87MG cell line (FIG. 29), consistent withprevious reports demonstrating that these cells express the wt EGFR(16). mAb 806 had binding levels similar to the control antibody,clearly demonstrating that it is unable to bind the wt EGFR (FIG. 29).Binding of the isotype control antibody to the U87MG.Δ2-7 andU87MG.wtEGFR cell lines was similar to that observed for the U87 MGcells. mAb 806 stained U87 MG.D2-7 and U87 MG. wtEGFR cells, indicatingthat mAb 806 specifically recognized the de2-7 EGFR and a subset of theoverexpressed EGFR (FIG. 29). As expected, the 528 antibody stained boththe U87MG.Δ2-7 and U87MG.wtEGFR cell lines (FIG. 29). The intensity of528 antibody staining on U87MG.wtEGFR cells was much higher than mAb806, suggesting that mAb 806 only recognizes a portion of theoverexpressed EGFR. The mAb 806 reactivity observed with U87MG.wtEGFRcells is similar to that obtained with A431 cells, another cell linethat over expresses the wt EGFR.3

A Scatchard analysis was performed using U87MG.Δ2-7 and A431 cells todetermine the relative affinity and binding sites for mAb 806 on eachcell line. mAb 806 had an affinity for the de2-7 EGFR receptor of1.1×10⁹ _(M) ⁻¹ and recognized an average (three separate experiments)of 2.4×10⁵ binding sites/cell. In contrast, the affinity of mAb 806 forthe wt EGFR on A431 cells was only 9.5×10⁷ M⁻¹. Interestingly, mAb 806recognized 2.3×10⁵ binding sites on the surface of A431, which is some10-fold lower than the reported number of EGFR found in these cells. Toconfirm the number of EGFR on the surface of our A431 cells, weperformed a Scatchard analysis using ¹²⁵I-labeled 528 antibody. Asexpected, this antibody bound to approximately 2×10⁶ sites on thesurface of A431 cells. Thus, it appears that mAb 806 only binds aportion of the EGFR receptors on the surface of A431 cells. Importantly,¹²⁵I-labeled mAb 806 did not bind to the parental U87 MG cells at all,even when the number of cells was increased to 1×10⁷.

Immunoprecipitations.

We further characterized mAb 806 reactivity in the various cell lines byimmunoprecipitation after 35 S-labeling using mAb 806, sc-03 (acommercial polyclonal antibody specific for the COOH-terminal domain ofthe EGFR) and a IgG2b isotype control. The sc-03 antibodyimmunoprecipitated three bands from U87MG.Δ2-7 cells, a doubletcorresponding to the two de2-7 EGFR bands observed in these cells and ahigher molecular weight band corresponding to the wt EGFR (FIG. 30). Incontrast, although mAb 806 immunoprecipitated the two de2-7 EGFR bands,the wt EGFR was completely absent (FIG. 30). The pattern seen inU87MG.wtEGFR and A431 cells was essentially identical. The sc-03antibody immunoprecipitated a single band corresponding to the wt EGFRfrom both cell lines (FIG. 30). The mAb 806 also immunoprecipitated asingle band corresponding to the wt EGFR from both U87MG.wtEGFR and A431cells (FIG. 30). Consistent with the FACS and Scatchard data, the amountof EGFR immunoprecipitated by mAb 806 was substantially less than thetotal EGFR present on the cell surface. Given that mAb 806 and the sc-03immunoprecipitated similar amounts of the de2-7 EGFR, this resultsupports the notion that the mAb 806 antibody only recognizes a portionof the EGFR in cells overexpressing the receptor. Comparisons betweenmAb 806 and the 528 antibody showed an identical pattern of reactivity(data not shown). An irrelevant IgG2b (an isotype control for mAb 806)did not immunoprecipitate EGFR from any of the cell lines (FIG. 30).Using identical conditions, mAb 806 did not immunoprecipitate the EGFRfrom the parental U87MG cells (data not shown).

Efficacy of mAb 806 in Preventative Models.

mAb 806 was examined for efficacy against U87MG and U87MG.Δ2-7 tumors ina preventative xenograft model. Antibody or vehicle was administeredi.p. the day before tumor inoculation and was given three times per weekfor 2 weeks (see “Materials and Methods”). At a dose of 1 mg/injection,mAb 806 had no effect on the growth of parental U87MG xenografts thatexpress the wt EGFR (FIG. 9A). In contrast, mAb 806 inhibitedsignificantly the growth of U87MG.Δ2-7 xenografts in a dose-dependentmanner (FIG. 9B). Twenty days after tumor inocu-lation, when controlanimals were sacrificed, the mean tumor volume was 1600±180 mm³ for thecontrol group, a significantly smaller 500±95 mm³ for the 0.1mg/injection group (P<0.0001) and 200±42 mm³ for the 1 mg/injectiongroup (P<0.0001). Treatment groups were sacrificed at day 24, at whichtime the mean tumor volumes were 1300±240 mm 3 for the 0.1 mg treatedgroup and 500±100 mm³ for the 1 mg group (P<0.005).

Efficacy of mAb 806 in Established Xenograft Models.

Given the efficacy of mAb 806 in the preventative xenograft model, itsability to inhibit the growth of established tumor xenografts wasexamined Antibody treatment was as described in the preventative model,except that it commenced when tumors had reached a mean tumor volume of65 mm³ (10 days after implantation) for the U87MG.Δ2-7 xenografts and 84mm³ (19 days after implantation) for the parental U87MG xenografts. Onceagain, mAb 806 had no effect on the growth of parental U87MG xenografts,even at a dose of 1 mg/injection (FIG. 10A). In contrast, mAb 806significantly inhibited the growth of U87MG.Δ2-7 xenografts in adose-dependent manner (FIG. 10B). At day 17, 1 day before controlanimals were sacrificed, the mean tumor volume was 900±200 mm³ for thecontrol group, 400±60 mm³ for the 0.1 mg/injection group (P<0.01), and220±60 mm³ for the 1 mg/injection group (P<0.002). Treatment ofU87MG.Δ2-7 xenografts with an IgG2b isotype control had no effect ontumor growth (data not shown).

To examine whether the growth inhibition observed with mAb 806 wasrestricted to cells expressing de2-7 EGFR, its efficacy against theU87MG.wtEGFR xenografts was also examined in an established model. Thesecells serve as a model for tumors containing amplification of the EGFRgene without de2-7 EGFR expression. mAb 806 treatment commenced whentumors had reached a mean tumor volume of 73 mm 3 (22 days afterimplantation). mAb 806 significantly inhibited the growth of establishedU87MG.wtEGFR xenografts when compared with control tumors treated withvehicle (FIG. 10C). On the day control animals were sacrificed, the meantumor volume was 1000±300 mm³ for the control group and 500±80 mm³ forthe group treated with 1 mg/injection (P<0.04).

Histological and Immunohistochemical Analysis of Established Tumors.

To evaluate potential histological differences between mAb 806-treatedand control U87MG.Δ2-7 and U87MG.wtEGFR xenografts, formalin-fixed,paraffin-embedded sections were stained with H&E (FIG. 31). Areas ofnecrosis were seen in sections from mAb 806-treated U87MG.Δ2-7 (mAb806-treated xenografts were collected 24 days after tumor inoculationand vehicle treated xenografts at 18 days), and U87MG.wtEGFR xenografts(mAb 806 xenografts were collected 42 days after tumor inoculation andvehicle treated xenografts at 37 days; FIG. 31). This result wasconsistently observed in a number of tumor xenografts (n 5 4 for eachcell line). However, sections from U87MG.Δ2-7 and U87MG.wtEGFRxenografts treated with vehicle (ii 5 5) did not display the same areasof necrosis seen after mAb 806 treatment (FIG. 31). Vehicle and mAb806-treated xenografts removed at identical times also showed thesedifferences in tumor necrosis (data not shown). Thus, the increase innecrosis observed was not caused by the longer growth periods used forthe mAb 806-treated xenografts. Furthermore, sections from mAb806-treated U87MG xenografts were also stained with H&E and did notreveal any areas of necrosis (data not shown), further supporting thehypothesis that mAb 806 binding induces decreased cell viability,resulting in increased necrosis within tumor xenografts.

An immunohistochemical analysis of U87MG, U87MG.Δ2-7, and U87MG.wtEGFRxenograft sections was performed to determine the levels of de2-7 and wtEGFR expression after mAb 806 treatment (FIG. 32). As expected, the 528antibody stained all xenografts sections with no obvious decrease inintensity between treated and control tumors (FIG. 32). Staining ofU87MG sections was undetectable with the mAb 806; however, positivestaining of U87MG.Δ2-7 and U87MG.wtEGFR xenograft sections was observed(FIG. 32). There was no difference in mAb 806 staining intensity betweencontrol and treated U87MG.Δ2-7 and U87MG.wtEGFR xenografts, suggestingthat antibody treatment does not lead to the selection of clonalvariants lacking mAb 806 reactivity.

Treatment of A431 Xenografts with mAb 806.

To demonstrate that the antitumor effects of mAb 806 were not restrictedto U87MG cells, the antibody was administrated to mice containing A431xenografts. These cells contain an amplified EGFR gene and expressapproximately 2×10⁶ receptors/cells. We have previously shown that mAb806 binds ˜10% of these EGFRs and targets A431 xenografts. (3) mAb 806significantly inhibited the growth of A431 xenografts when examined inthe preventative xenograft model described previously (FIG. 11A). At day13, when control animals were sacrificed, the mean tumor volume was1400±150 mm³ in the vehicle-treated group and 260±60 mm³ for the 1mg/injection treatment group (P<0.0001). In a separate experiment, adose of 0.1 mg of mAb also inhibited significantly (P<0.05) the growthof A431 xenografts in a preventative model (data not shown).

Given the efficacy of mAb 806 in the preventative A431 xenograft model,its ability to inhibit the growth of established tumor xenografts wasexamined Antibody treatment was as described in the preventative model,except it was not started until tumors had reached a mean tumor volumeof 200±20 mm³ mAb 806 significantly inhibited the growth of establishedA431 xenografts (FIG. 11B). At day 13, the day control animals weresacrificed, the mean tumor volume was 1100±100 mm³ for the control groupand 450±70 mm³ for the 1 mg/injection group (P<0.0001).

We have shown previously³ that mAb 806 targets both de2-7EGFR-transfected U87MG xenografts and A431 xenografts that over expressthe wt EGFR. mAb 806 did not target parental U87MG cells, which express˜10⁵ EGFR³ (16). As assessed by FACS, immunohistochemistry, andimmunoprecipitation, we now demonstrate that mAb 806 is also able tospecifically bind U87MG.wtEGFR cells, which express >10⁶ EGFRs/cell.Thus, the previous observed binding of mAb 806 to A431 cells is not theresult of some unusual property of these cells but rather appears to bea more general phenomenon related to over expression of the wt EGFR.

(a) We were unable to detect mAb 806 binding to the parental U87MGcellline, which expresses 1×10⁵ wt EGFRs/cell (16), either by FACS,immunoprecipitation, immunohistochemistry, or with iodinated antibody.Indeed, iodinated mAb 806 did not bind to U87 MG cell pellets containing1×10⁷ cells, which based on the Scatchard data using 1×10⁶ A431 cells,are conditions that should detect low level antibody binding (i.e., thetotal number of receptors being similar in both cases).

(b) Scatchard analysis clearly showed that mAb 806 only bound to 10% ofthe total EGFR on the surface of A431 cells. If mAb 806 simply binds tothe wt EGFR with low affinity, then it should have bound to aconsiderably higher percentage of the receptor.

(c) Comparative immunoprecipitation of the A431 and U87MG. wtEGFR celllines with mAb 806 and the sc-03 antibody also supported the hypothesisthat only a subset of receptors are recognized by mAb 806. Takentogether, these results support the notion that mAb 806 recognizes aEGFR subset on the surface of cells overexpressing the EGFR. We arecurrently analyzing the EGFR immunoprecipitated by mAb 806 to see if itdisplays altered biochemical properties related to glycosylation orkinase activity.

The xenograft studies with mAb 806 described here demonstratedose-dependent inhibition of U87MG.Δ2-7 xenograft growth. In contrast,no inhibition of parental U87 MG xenografts was observed, despite thefact that they continue to express the wt EGFR in vivo. mAb 806 not onlysignificantly reduced xenograft volume, it also induced significantnecrosis within the tumor. As noted above, other de2-7 EGFR-specificmAbs have been generated (20-22), but this is the first report showingthe successful therapeutic use of such an antibody in vivo against ahuman de2-7 EGFR-expressing glioma xenograft. A recent reportdemonstrated that the de2-7 EGFR-specific Y10 mAb had in vivo antitumoractivity against murine B16 melanoma cells transfected with a murinehomologue of the human de2-7 EGFR (33). Y10 mediated in vitro cell lysis(>90%) of B16 melanoma cells expressing the de2-7 EGFR in the absence ofcomplement or effector cells. In contrast to their in vitroobservations, the in vivo Y10 antibody efficacy was completely mediatedthrough Fc function when using B16 melanoma cells grown as xenografts inan immunocompetent model. Thus, the direct effects observed in vitro donot seem to be replicated when cells are grown as tumor xenografts.

Overexpression of the EGFR has been reported in a number of differenttumors and is observed in most gliomas (4, 14). It has been proposedthat the subsequent EGFR over expression mediated by receptor geneamplification may confer a growth advantage by increasing intracellularsignaling and cell growth (34). The U87MG cell line was transfected withthe wt EGFR to produce a glioma cell that mimics the process of EGFRgene amplification. Treatment of established U87MG.wtEGFR xenograftswith mAb 806 resulted in significant growth inhibition. Thus, mAb 806also mediates in vivo antitumor activity against cells overexpressingthe EGFR. Interestingly, mAb 806 inhibition of U87MG.wtEGFR xenograftswas less pronounced than that observed with U87MG.Δ2-7 tumors. Thisprobably reflects the fact that mAb 806 has a lower affinity for theoverexpressed wt EGFR and only binds a small proportion of receptorsexpressed on the cell surface. (3) However, it should be noted thatdespite the small effect on U87MG.wtEGFR xenograft volumes, mAb 806treatment produced large areas of necrosis within these xenografts. Toexclude the possibility that mAb 806 only mediates inhibition of theU87MG-derived cell lines, we tested its efficacy against A431xenografts. This squamous cell carcinoma-derived cell line containssignificant EGFR gene amplification, which is retained both in vitro andin vivo. Treatment of A431 xenografts with mAb 806 produced significantgrowth inhibition in both a preventative and established model,indicating the antitumor effects of mAb 806 are not restricted totransfected U87MG cell lines.

Complete prevention of A431 xenograft growth by antibody treatment hasbeen reported previously. The wt EGFR mAbs 528, 225, and 425 allprevented the formation of A431 xenografts when administered either onthe day or 1 day after tumor inoculation (9, 10). The reason for thisdifference in efficacy between these wt EGFR anti-bodies and mAb 806 isnot known but may be related to the mechanism of cell growth inhibition.The wt EGFR antibodies function by blocking ligand binding to the EGFR,but this is probably not the case with mAb 806 because it only binds asmall EGFR subset on the surface of A431 cells. The significant efficacyof mAb 806 against U87MG cells expressing the ligand-independent de2-7EGFR further supports the notion that this antibody mediates itsantitumor activity by a mechanism not involving ligand blockade.Therefore, we are currently investigating the non-immunological andimmunological mechanisms that contribute to the antitumor effects of mAb806. Non-immunological mechanisms may include subtle changes in receptorlevels, blockade of signaling, or induction of inappropriate signaling.

Previously, agents such as doxorubicin and cisplatin in conjunction withwt EGFR antibodies have produced enhanced antitumor activity (35, 36).The combination of doxorubicin and mAb 528 resulted in total eradicationof established A431 xenografts, whereas treatment with either agentalone caused only temporary in vivo growth inhibition (36). Likewise,the combination of cisplatin and either mAb 528 or 225 also led to theeradication of well-established A431 xenografts, which was not observedwhen treatment with either agent was used (35). Thus, future studiesinvolving the combination of chemotherapeutic agents with mAb 806 areplanned using xenograft models.

Maybe the most important advantage of mAb 806 compared with current EGFRantibodies is that it should be possible to directly conjugate cytotoxicagents to mAb 806. This approach is not feasible with currentEGFR-specific antibodies because they target the liver and cytotoxicconjugation would almost certainly induce severe toxicity. Conjugationof cytotoxic agents such as drugs (37) or radioisotopes (38) toantibodies has the potential to improve efficacy and reduce the systemictoxicity of these agents. This study clearly demonstrates that mAb 806has significant in vivo antitumor activity against de2-7 EGFR-positivexenografts and tumors overexpressing the EGFR. The unique specificity ofmAb 806 suggests immunotherapeutic potential in targeting a number oftumor types, particularly head and neck tumors and glioma, without therestrictions associated with normal tissue uptake.

References

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Therapeutic efficacy of anti-Lewis(y) humanized    3S193 radioimmunotherapy in a breast cancer model: enhanced activity    when combined with Taxol chemotherapy. Clin. Cancer Res., 6:    3621-3628, 2000.-   27. Atlas, I., Mendelsohn, J., Baselga, J., Fair, W. R., Masui, H.,    and Kumar, R. Growth regulation of human renal carcinoma cells: role    of transforming growth factor a. Cancer Res., 52: 3335-3339, 1992.-   28. Perez-Soler, R., Donato, N.J., Shin, D. M., Rosenblum, M. G.,    Zhang, H. Z., Tornos, C., Brewer, H., Chan, J. C., Lee, J. S.,    Hong, W. K., et al. Tumor epidermal growth factor receptor studies    in patients with non-small-cell lung cancer or head and neck cancer    treated with monoclonal antibody RG 83852. J. Clin. Oncol., 12:    730-739, 1994.-   29. Wersall, P., Ohlsson, I., Biberfeld, P., Collins, V. P., von    Krusenstjerna, S., Larsson, S., Mellstedt, H., and Boethius, J.    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EXAMPLE 21 Construction, Expression and Analysis of Chimeric 806Antibody

Chimeric antibodies are a class of molecules in which heavy and lightchain variable regions of for instance, a mouse, rat or other speciesare joined onto human heavy and light chain regions. Chimeric antibodiesare produced recombinantly. One advantage of chimeric antibodies is thatthey can reduce xenoantigenic effects, the inherent immunogenicity ofnon-human antibodies (for instance, mouse, rat or other species). Inaddition, recombinantly prepared chimeric antibodies can often beproduced in large quantities, particularly when utilizing high levelexpression vectors.

For high level production, the most widely used mammalian expressionsystem is one which utilizes the gene amplification procedure offered bydehydrofolate reductase deficient (“dhfr−”) Chinese hamster ovary cells.The system is well known to the skilled artisan. The system is basedupon the dehydrofolate reductase “dhfr” gene, which encodes the DHFRenzyme, which catalyzes conversion of dehydrofolate to tetrahydrofolate.In order to achieve high production, dhfr-CHO cells are transfected withan expression vector containing a functional DHFR gene, together with agene that encodes a desired protein. In this case, the desired proteinis recombinant antibody heavy chain and/or light chain.

By increasing the amount of the competitive DHFR inhibitor methotrexate(MTX), the recombinant cells develop resistance by amplifying the dhfrgene. In standard cases, the amplification unit employed is much largerthan the size of the dhfr gene, and as a result the antibody heavy chainis co-amplified.

When large scale production of the protein, such as the antibody chain,is desired, both the expression level, and the stability of the cellsbeing employed, are critical. In long term culture, recombinant CHO cellpopulations lose homogeneity with respect to their specific antibodyproductivity during amplification, even though they derive from asingle, parental clone.

Bicistronic expression vectors were prepared for use in recombinantexpression of the chimeric antibodies. These bicistronic expressionvectors, employ an “internal ribosomal entry site” or “IRES.” In theseconstructs for production of chimericanti-EGFR, the immunoglobulinchains and selectable markers cDNAs are linked via an IRES. IRES arecis-acting elements that recruit the small ribosomal subunits to aninternal initiator codon in the mRNA with the help of cellulartrans-acting factors. IRES facilitate the expression of two or moreproteins from a polycistronic transcription unit in eukaryotic cells.The use of bicistronic expression vectors in which the selectable markergene is translated in a cap dependent manner, and the gene of interestin an IRES dependent manner has been applied to a variety ofexperimental methods. IRES elements have been successfully incorporatedinto vectors for cellular transformation, production of transgenicanimals, recombinant protein production, gene therapy, gene trapping,and gene targeting.

Synopsis of Chimeric Antibody 806 (ch806) Construction

The chimeric 806 antibody was generated by cloning the VH and VL of the806 antibody from the parental murine hybridoma using standard molecularbiology techniques. The VH and VL were then cloned into the pRENmammalian expression vectors, the construction of which are set forth inTable 6 and Table 7 and transfected into CHO (DHFR−/−ve) cells foramplification and expression. Briefly, following trypsinization 4×10⁶CHO cells were co-transferred with 10

|g of each of the LC and HC expression vectors using electroporationunder standard conditions. Following a 10 min rest period at roomtemperature, the cells were added to 15 ml medium (10% fetal calf serum,hypoxanthine/thymidine supplement with additives) and transferred to15×10 cm cell culture petri dishes. The plates were then placed into theincubator under normal conditions for 2 days. At this point, theaddition of gentamycin, 5 nM methotrexate, the replacement of fetal calfserum with dialyzed fetal calf serum and the removal ofhypoxanthine/thymidine, initiated the selection for clones that weresuccessfully transfected with both the LC and HC from the medium. At day17 following transfection, individual clones growing under selectionwere picked and screened for expression of the chimeric 806 antibody. AnELISA was utilized for screening and consisted of coating an ELISA platewith denatured soluble EGF receptor (denatured EGFR is known to allow806 binding). This assay allows for the screening of production levelsby individual clones and also for the functionality of the antibodybeing screened. All clones were shown to be producing functional ch806and the best producer was taken and expanded for amplification. Toamplify the level of ch806 being produced, the highest producing clonewas subjected to reselection under a higher methotrexate concentration(100 nM vs 5 nM). This was undertaken using the aforementionedprocedures.

Clones growing at 100 nM MTX were then passed onto the BiologicalProduction Facility, Ludwig Institute, Melbourne, Australia formeasurement of production levels, weaning off serum, cell banking. Thecell line has been shown to stably produce ˜10 mg/liter in rollerbottles.

The nucleic acid sequence of the pREN ch806 LC neo vector is provided inSEQ ID NO:7 and the corresponding amino acid sequence is defined in SEQID NO:9. The nucleic acid sequence of the pREN ch806 HC DHFR vector isprovided in SEQ ID NO:8 and the corresponding amino acid sequence is setforth in SEQ ID NO:10.

FIG. 33 depicts the vectors pREN-HC and pREN-LC, which employ an IRES.The pREN bicistronic vector system is described and disclosed inco-pending U.S. Ser. No. 60/355,838 filed Feb. 13, 2002, which isincorporated herein by reference in its entirety.

Ch806 was assessed by FACS analysis to demonstrate that the chimeric 806displays identical binding specificity to that of the murine parentalantibody. Analysis was performed using wild type cells (U87MG parentalcells), cells overexpressing the EGF receptor (A431 cells and UA87 wtEGFR cells) and UA87 Δ2-7 cells (data not shown). Similar bindingspecificity of Mab806 and ch806 was obtained using cells overexpressingEGFR and cells expressing the de2-7 EGFR. No binding was observed inwild type cells. Scatchard analysis revealed a binding affinity forradiolabeled ch806 of 6.4×10⁹ M⁻¹ using U87MG.de2-7 cells (data notshown).

Biodistribution analysis of the ch806 antibody was performed in BALB/cnude mice bearing U87MG-de2-7 xenograft tumors and the results are shownin FIG. 34. Mice were injected with 5 μg of radiolabelled antibody andwere sacrificed in groups of four per time point at 8, 24, 48 and 74hours. Organs were collected, weighed and radioactivity measured in agamma counter. ¹²⁵I-labelled ch806 displays reduced targeting to thetumor compared to ¹¹¹In-labelled ch806, which has high tumor uptake andcumulative tumor retention over the 74 hour time period. At 74 hours,the ¹¹¹In-labelled antibody displays approximately 30% ID/gram tissueand a tumor to blood ratio of 4.0 (FIG. 35). The ¹¹¹In-labelled ch806shows some nonspecific retention in the liver, spleen and kidneys. Thisis common for the use of this isotope and decreases with time, whichsupports that this binding is non-specific to ch806 and due to ¹¹¹Inbinding.

Chimeric antibody ch806 was assessed for therapeutic efficacy in anestablished tumor model. 3×10⁶ U87MG.Δ2-7 cells in 100 μl of PBS wereinoculated s.c. into both flanks of 4-6 week old female nude mice(Animal Research Center, Western Australia, Australia). The mAb806 wasincluded as a positive control. The results are depicted in FIG. 36.Treatment was started when tumors had reached a mean volume of 50 mm³and consisted of 1 mg of ch806 or mAb806 given i.p. for a total of 5injections on the days indicated. Tumor volume in mm³ was determinedusing the formula (length×width²)/2, where length was the longest axisand width the measurement at right angles to the length. Data wasexpressed as mean tumor volume+/−S.E. for each treatment group. Thech806 and mAb806 displayed nearly identical anti-tumor activity againstU87MG.Δ2-7 xenografts.

Analysis of Ch806 Immune Effector Function

Materials and Methods

Antibodies and Cell Lines:

Murine anti-de2-7 EGFR monoclonal mAb 806, chimeric antibody ch806(IgG₁) and control isotype matched chimeric anti-G250 monoclonalantibody cG250 were prepared by the Biological Production Facility,Ludwig Institute for Cancer Research, Melbourne, Australia. Bothcomplement-dependant cytotoxicity (CDC) and antibody-dependentcellular-cytotoxicity (ADCC) assays utilised U87MG.de2-7 and A431 cellsas target cells. The previously described U87MG.de2-7 cell line is ahuman astrocytoma cell line infected with a retrovirus containing thede2-7EGFR (Nishikawa, R. et al. (1994) Proc Natl Acad Sci USA. 91:7727-31). Human squamous carcinoma A431 cells were purchased from theAmerican Type Culture Collection (Manassas, Va.). All cell lines werecultured in DMEM/F-12 with GLUTAMAX™ (Life Technologies, Melbourne,Australia) supplemented with 10% heat-inactivated FCS(CSL, Melbourne,Australia), 100 units/ml penicillin and 100 ng/ml streptomycin. Tomaintain selection for retrovirally transfected U87MG.de2-7 cells, 400ng/ml G418 was included in the media.

Preparation of human peripheral blood mononuclear cells (PBMC) EffectorCells: PBMCs were isolated from healthy volunteer donor blood.Heparinised whole blood was fractionated by density centrifugation onFicoll-Hypaque (ICN Biomedical Inc., Ohio, USA). PBMC fractions wascollected and washed three times with RPML⁺ 1640 supplemented with 100U/ml penicillin and 100 μg/ml streptomycin, 2 mM L-glutamine, containing5% heat-inactivated FCS.

Preparation of Target Cells:

CDC and ADCC assays were performed by a modification of a previouslypublished method ((Nelson, D. L. et al. (1991) In: J. E. Colignan, A. M.Kruisbeek, D. D. Margulies, E. M. Shevach, and W. Strober (eds.),Current Protocols in Immunology, pp. 7.27.1. New York: Greene PublishingWiley Interscience). Briefly, 5×10⁶ target U87MG.de2-7 and A431 cellswere labeled with 50 μCi ⁵¹Cr (Geneworks, Adelaide, Australia) per 1×10⁶cells and incubated for 2 hr at 37° C. The cells were then washed threetime with PBS (0.05M, pH 7.4) and a fourth wash with culture medium.Aliquots (1×10⁴ cells/50 ul) of the labeled cells were added to eachwell of 96-well microtitre plates (NUNC, Roskilde, Denmark).

CDC Assay:

To 50 μl labeled target cells, 50 μl ch806 or isotype control antibodycG250 were added in triplicate over the concentration range 0.00315-10μg/ml, and incubated on ice 5 min. Fifty μl of freshly prepared healthydonor complement (serum) was then added to yield a 1:3 final dilution ofthe serum. The microtitre plates were incubated for 4 hr at 37° C.Following centrifugation, the released ⁵¹Cr in the supernatant wascounted (COBRA II™ automated Gamma Counter, Can berra Packard,Melbourne, Australia). Percentage specific lysis was calculated from theexperimental ⁵¹Cr release, the total (50 μl target cells+100 μl 10%TWEEN™ 20 and spontaneous (50 μl target cells+100 μl medium) release.

ADCC Assay:

Ch806-mediated ADCC effected by healthy donor PBMCs was measured by two4-hr ⁵¹Cr release assays. In the first assay, labelled target cells wereplated with the effector cells in 96-well “U” bottom microplates (NUNC,Roskilde, Denmark) at effector/target (E:T) cell ratios of 50:1. ForADCC activity measurements, 0.00315-10 μg/mg (final concentration) testand control antibodies were added in triplicate to each well. In thesecond ADCC assay, the ADCC activity of ch806 was compared with theparental murine mAb 806 over a range of Effector: Target cell ratioswith the test antibody concentration constant at 1 ug/ml. In bothassays, micotitre plates were incubated at 37° C. for 4 hours, then 50ul supernatant was harvested from each well and released ⁵¹Cr wasdetermined by gamma counting (COBRA II™ automated Gamma Counter, Canberra Packard, Melbourne, Australia). Controls included in the assayscorrected for spontaneous release (medium alone) and total release (10%TWEEN™ 20/PBS). Appropriate controls with the same subclass antibodywere run in parallel.

The percentage cell lysis (cytotoxicity) was calculated according to theformula:

${{Percentage}\mspace{14mu}{Cytotoxicity}} = {\frac{{{Sample}\mspace{14mu}{Counts}} - {{Spontaneous}\mspace{14mu}{Release}}}{{{Total}\mspace{14mu}{Release}} - {{Spontaneous}\mspace{14mu}{Release}}} \times 100}$

The percent (%) cytotoxicity was plotted versus concentration ofantibody (μg/ml).

Results

The results of the CDC analyses are presented in FIG. 37. Minimal CDCactivity was observed in the presence of up to 10 μg/ml ch806 with CDCcomparable to that observed with isotype control cG250.

Ch806 mediated ADCC on target U87MG.de2-7 and A431 cells at E:T ratio of50:1 is presented in FIG. 38. Effective ch806 specific cytotoxicity wasdisplayed against target U87MG.de2-7 cells, but minimal ADCC wasmediated by ch806 on A431 cells. The levels of cytotoxicity achievedreflect the number of ch806 binding sites on the two cell populations.Target U87MG.de2-7 cells express ˜1×10⁶ de2-7EGFR which are specificallyrecognised by ch806, while only a subset of the 1×10⁶ wild-type EGFRmolecules expressed on A431 cells are recognised by ch806 (see aboveExamples).

Further ADCC analyses were performed to compare the ADCC mediated by 1ug/ml ch806 on target U87MG.de2-7 cells with that effected by 1 μg/mlparental murine mAb 806. Results are presented in FIG. 39. Chimerisationof mAb 806 has effected marked improvement of the ADCC achieved by theparental murine mAb with greater than 30% cytotoxicity effected at E:Tratios 25:1 and 50:1.

The lack of parental murine mAb 806 immune effector function has beenmarkedly improved upon chimerisation. Ch806 mediates good ADCC, butminimal CDC activity.

EXAMPLE 22 Generation of Anti-Idiotype Antibodies to Chimeric Antibodych806

To assist the clinical evaluation of mAb806 or ch806, laboratory assaysare required to monitor the serum pharmacokinetics of the antibodies andquantitate any immune responses to the mouse-human chimeric antibody.Mouse monoclonal anti-idiotypic antibodies (anti-ids) were generated andcharacterised for suitability as ELISA reagents for measuring ch806 inpatient sera samples and use as positive controls in human anti-chimericantibody immune response analyses. These anti-idiotype antibodies mayalso be useful as therapeutic or prophylactic vaccines, generating anatural anti-EGFR antibody response in patients.

Methods for generating anti-idiotype antibodies are well known in theart (Bhattacharya-Chatterjee, Chatterjee et al. 2001, Uemura et al.1994, Steffens, Boerman et al. 1997, Safa and Foon 2001, Brown and Ling1988).

Mouse monoclonal anti-idiotypic antibodies (anti-ids) were, briefly,generated as follows. Splenocytes from mice immunized with ch806 werefused with SP2/0-AG14 plasmacytoma cells and antibody producinghybridomas were selected through ELISA for specific binding to ch806 andcompetitive binding for antigen (FIG. 40). Twenty-five hybridomas wereinitially selected and four, designated LMH-11, -12-13 and -14, secretedantibodies that demonstrated specific binding to ch806, mAb 806 and wereable to neutralise ch806 or mAb 806 antigen binding activity (FIG. 41).The recognition of the ch806/mAb 806 idiotope or CDR region wasdemonstrated by lack of cross-reactivity with purified polyclonal humanIgG.

In the absence of readily available recombinant antigen de2-7 EGFR toassist with the determination of ch806 in serum samples, the ability ofthe novel anti-idiotype ch806 antibodies to concurrently bind 806variable regions was exploited in the development of a sensitive,specific ELISA for measuring ch806 in clinical samples (FIG. 42).

Using LMH-12 for capture and Biotinylated-LMH-12 for detection, thevalidated ELISA demonstrated highly reproducible binding curves formeasuring ch806 (2 μg/ml-1.6 ng/ml) in sera with a 3 ng/ml limit ofdetection. (n=12; 1-100 ng/ml, Coefficient of Variation <25%; 100ng/ml-5 ug/ml, Coefficient of Variation <15%). No background binding wasevident with the three healthy donor sera tested and negligible bindingwas observed with isotype control hu3S193. The hybridoma produces highlevels of antibody LMH-12, and larger scale production is planned toenable the measurement of ch806 and quantitation of any immune responsesin clinical samples. (Brown and Ling 1988)

Results

Mice Immunization and Hybridoma Clone Selection

Immunoreactivity of pre- and post-immunization sera samples indicatedthe development of high titer mouse anti-ch806 and anti-huIgG mAbs.Twenty-five hybridomas producing antibodies that bound ch806, but nothuIgG, were initially selected. The binding characteristics of some ofthese hybridomas are shown in FIGS. 42A and 42B. Four of theseanti-ch806 hybridomas with high affinity binding (clones 3E3, 5B8, 9D6and 4D8) were subsequently pursued for clonal expansion from singlecells by limiting dilution and designated Ludwig Institute for CancerResearch Melbourne Hybridoma (LMH)-11, -12, -13, and -14, respectively(FIG. 42).

Binding and Blocking Activities of Selected Anti-Idiotype Antibodies

The ability of anti-ch806 antibodies to concurrently bind two ch806antibodies is a desirable feature for their use as reagents in an ELISAfor determining serum ch806 levels. Clonal hybridomas, LMH-11, -12, -13,and -14 demonstrated concurrent binding (data not shown).

After clonal expansion, the hybridoma culture supernatants were examinedby ELISA for the ability to neutralise ch806 or mAb 806 antigen bindingactivity with sEGFR621. Results demonstrated the antagonist activity ofanti-idiotype mAbs LMH-11, -12, -13, and -14 with the blocking insolution of both ch806 and murine mAb 806 binding to plates coated withsEGFR (FIG. 41 for LMH-11, -12, -13). Following larger scale culture inroller bottles the binding specificity's of the established clonalhybridomas, LMH-11, -12, -13, and -14 were verified by ELISA. LMH-11through -14 antibodies were identified as isotype IgG1κ by mousemonoclonal antibody isotyping kit.

ch806 in Clinical Serum Samples: Pharmacokinetic ELISA Assay Development

To assist with the determination of ch806 in serum samples, the abilityof the anti-idiotype ch806 antibodies to concurrently bind the 806variable region was exploited in the development of a sensitive andspecific ELISA assay for ch806 in clinical samples. The three purifiedclones LMH-11, -12, and -13 (FIGS. 49B and 49C, respectively) werecompared for their ability to capture and then detect bound ch806 insera. Results indicated using LMH-12 (10 μg/ml) for capture andbiotinylated-LMH-12 for detection yielded the highest sensitivity forch806 in serum (3 ng/ml) with negligible background binding.

Having established the optimal pharmacokinetic ELISA conditions using 1μg/ml anti-idiotype LMH-12 and 1 μg/ml biotinylated LMH-12 for captureand detection, respectively, validation of the method was performed.Three separate ELISAs were performed in quadruplicate to measure ch806in donor serum from three healthy donors or 1% BSA/media with isotypecontrol hu3S193. Results of the validation are presented in FIG. 43 anddemonstrate highly reproducible binding curves for measuring ch806 (2μg/ml-1.6 ng/ml) in sera with a 3 ng/ml limit of detection. (n=12; 1-100ng/ml, Coefficient of Variation <25%; 100 ng/ml-5 μg/ml, Coefficient ofVariation <15%). No background binding was evident with any of the threesera tested and negligible binding was observed with isotype controlhu3S193.

References

-   Brown, G. and N. Ling (1988). Murine Monoclonal Antibodies.    Antibodies, Volume I. A Practical Approach. D. Catty. Oxford,    England, IRL Press: 81-104.-   Bhattacharya-Chatterjee, M., S. K. Chatterjee, et al. (2001). “The    anti-idiotype vaccines for immunotherapy.” Curr Opin Mol Ther 3(1):    63-9.-   Domagala, T., N. Konstantopoulos, et al. (2000). “Stoichiometry,    kinetic and binding analysis of the interaction between epidermal    growth factor (EGF) and the extracellular domain of the EGF    receptor.” Growth Factors 18(1): 11-29.-   Safa, M. M. and K. A. Foon (2001). “Adjuvant immunotherapy for    melanoma and colorectal cancers.” Semin Oncol 28(1): 68-92.-   Uemura, H., E. Okajima, et al. (1994). “Internal image anti-idiotype    antibodies related to renal-cell carcinoma-associated antigen G250.”    Int J Cancer 56(4): 609-14.

EXAMPLE 23 Assessment of Carbohydrate Structures and AntibodyRecognition

Experiments were undertaken to further assess the role of carbohydratestructures in the binding and recognition of the EGFR, both amplifiedand de2-7 EGFR, by the mAb806 antibody.

To determine if carbohydrate structures are directly involved in the mAb806 epitope, the recombinant sEGFR expressed in CHO cells was treatedwith PNGase F to remove N-linked glycosylation. Following treatment, theprotein was run on SDS-PAGE, transferred to membrane and immunoblottedwith mAb 806 (FIG. 44). As expected, the deglycosylated sEGFR ran fasteron SDS-PAGE, indicating that the carbohydrates had been successfullyremoved. The mAb 806 antibody clearly bound the deglycosylated materialdemonstrating the antibody epitope is peptide in nature and not solely aglycosylation epitope.

Lysates, prepared from cell lines metabolically labelled with ³⁵S, wereimmunoprecipitated with different antibodies directed to the EGFR (FIG.45). As expected, the 528 antibody immunoprecipitated 3 bands fromU87MG.Δ2-7 cells, an upper band corresponding to the wild type (wt) EGFRand two lower bands corresponding to the de2-7 EGFR. These two de2-7EGFR bands have been reported previously and are assumed to representdifferential glycosylation (Chu et al. (1997) Biochem. J. June 15; 324(Pt 3):885-861). In contrast, mAb 806 only immunoprecipitated the twode2-7 EGFR bands, with the wt receptor being completely absent evenafter over-exposure (data not shown). Interestingly, mAb806 showedincreased relative reactivity with the lower de2-7 EGFR band butdecreased reactivity with the upper band when compared to the 528antibody. The SC-03 antibody, a commercial rabbit polyclonal antibodydirected to C-terminal domain of the EGFR, immunoprecipitated the threeEGFR bands as seen with the 528 antibody although the total amount ofreceptor immunoprecipitated by this antibody was considerably less. Nobands were observed when using an irrelevant IgG2b antibody as a controlfor mAb 806.

The 528 antibody immunoprecipitated a single band from U87MG.wtEGFRcells corresponding to the wt receptor (FIG. 45). MAb 806 alsoimmunoprecipitated a single band from these cells, however, this EGFRband clearly migrated faster than the 528 reactive receptor. The SC-03antibody immunoprecipitated both EGFR reactive bands from U87MG.wtEGFRcells, further confirming that the mAb 806 and 528 recognize differentforms of the EGFR in whole cell lysates from these cells.

As observed with U87MG.wtEGFR cells, the 528 antibody immunoprecipitateda single EGFR band from A431 cells (FIG. 45). The 528 reactive EGFR bandis very broad on these low percentage gels (6%) and probably reflectsthe diversity of receptor glycosylation. A single EGFR band was alsoseen following immunoprecipitation with mAb 806. While this EGFR banddid not migrate considerably faster than the 528 overall broad reactiveband, it was located at the leading edge of the broad 528 band in areproducible fashion. Unlike U87MG.Δ2-7 cell lysates, the total amountof EGFR immunoprecipitated by mAb 806 from A431 lysates was considerablyless than with the 528 antibody, a result consistent with our Scatcharddata showing mAb 806 only recognizes a portion of the EGFR on thesurface of these cells (see Example 4 above) Immunoprecipitation withSC-03 resulted in a single broad EGFR band as for the 528 antibody.Similar results were obtained with HN5 cells (data not shown). Takentogether, this data indicates that mAb 806 preferentially reacts withfaster migrating species of the EGFR, which may represent differentiallyglycosylated forms of the receptor.

In order to determine at what stage of receptor processing mAb 806reactivity appeared a pulse/chase experiment was conducted. A431 andU87MG.Δ2-7 cells were pulsed for 5 min with ³⁵S methionine/cysteine,then incubated at 37° C. for various times before immunoprecipitationwith mAb 806 or 528 (FIG. 46). The immunoprecipitation pattern in A431cells with the 528 antibody was typical for a conformational dependentantibody specific for the EGFR. A small amount of receptor wasimmunoprecipitated at 0 min (i e after 5 min pulse) with the amount oflabelled EGFR increasing at each time point. There was also a concurrentincrease in the molecular weight of the receptor with time. In contrast,the mAb 806 reactive EGFR material was present at high levels at 0 min,peaked at 20 min and then reduced at each further time point. Thus, itappears that mAb 806 preferentially recognizes a form of the EGFR foundat an early stage of processing.

The antibody reactivity observed in pulse-labelled U87MG.Δ2-7 cells wasmore complicated. Immunoprecipitation with the 528 antibody at 0 minrevealed that a small amount of the lower de2-7 EGFR band was labelled(FIG. 46). The amount of 528 reactive de2-7 EGFR lower band increasedwith time, peaking at 60 min and declining slowly at 2 and 4 h. Nosignificant amount of the labelled upper band of de2-7 EGFR was detecteduntil 60 min, after which the level continued to increase until the endof the time course. This clearly indicates that the upper de2-7 EGFR isa more mature form of the receptor. MAb 806 reactivity also variedduring the time course study, however mAb 806 preferentiallyprecipitated the lower band of the de2-7 EGFR. Indeed, there were nosignificant levels of mAb 806 upper band seen until 4 h after labelling.

The above experiments suggest that mab 806 preferentially reacts with amore immature glycosylation form of the de2-7 and wt EGFR. Thispossibility was tested by immunoprecipitating the EGFR from differentcells lines labelled overnight with ³⁵S methionine/cysteine and thensubjecting the resultant precipitates to Endoglycosidase H (Endo H)digestion. This enzyme preferentially removes high mannose typecarbohydrates (i.e. immature glycosylation) from proteins while leavingcomplex carbohydrates (i.e. matureglycosylation) intact.Immunoprecipitation and digestion with Endo H of labelled U87MG.Δ2-7cell lysates with 528, mAb 806 and SC-03 gave similar results (FIG. 47).As predicted, the lower de2-7 EGFR band was fully sensitive to Endo Hdigestion, migrating faster on SDS-PAGE after Endo H digestion,demonstrating that this band represents the high mannose form of thede2-7 EGFR. The upper de2-7 EGFR band was essentially resistant to EndoH digestion, showing only a very slight difference in migration afterEndo H digestion, indicating that the majority of the carbohydratestructures are of the complex type. The small but reproducible decreasein the molecular weight of the upper band following enzyme digestionsuggests that while the carbohydrates on the upper de2-7 EGFR band arepredominantly of the complex type, it does possess some high mannosestructures. Interestingly, these cells also express low amounts ofendogenous wt EGFR that is clearly visible following 528immunoprecipitation. There was also a small but noticeable reduction inmolecular weight of the wt receptor following Endo H digestion,indicating that it also contains high mannose structures.

The sensitivity of the immunoprecipitated wt EGFR to Endo H digestionwas similar in both U87MG.wtEGFR and A431 cells (FIG. 47). The bulk ofthe material precipitated by the 528 antibody was resistant to the EndoH enzyme although a small amount of the material was of the high mannoseform. Once again there was a small decrease in the molecular weight ofthe wt EGFR following Endo H digestion suggesting that it does containsome high mannose structures. The results using the SC-03 antibody weresimilar to the 528 antibody. In contrast, the majority of the EGFRprecipitated by mAb 806 was sensitive to Endo H in both U87MG.wtEGFR andA431 cells, confirming that mAb 806 preferentially recognizes the highmannose form of the EGFR. Similar results were obtained with HN-5 cells,wherein the majority of the material precipitated by mAb 806 wassensitive to Endo H digestion, while the majority of the materialprecipitated by mAb528 and SC-03 was resistant to Endo H digestion (datanot shown).

Cell surface iodination of the A431 cell line, was performed with ¹²⁵Ifollowed by immunoprecipitation with the 806 antibody. The protocol forsurface iodination was as follows: The cell lysis, immunoprecipitation,Endo H digestion, SDS PAGE and autoradiography are as described aboveherein. For labeling, cells were grown in media with 10% FCS, detachedwith EDTA, washed twice with PBS then resuspended in 400 ul of PBS(approx 2-3×10⁶ cells). To this was added 15 ul of ¹²⁵I (100 mCi/mlstock), 100 ul bovine lactoperoxidase (1 mg/ml) stock, 10 ul H₂O₂ (0.1%stock) and this was incubated for 5 min. A further 10 ul of H₂O₂ wasthen added and the incubation continued for a further 3 min. Cells werethen washed again 3 times with PBS and lysed in 1% TRITON™. Cell surfaceiodination of the A431 cell line with lactoperoxidase, followed byimmunoprecipitation with the 806 antibody, showed that, similar to thewhole cell lysates described above, the predominant form of the EGFRrecognized by 806 bound on the cell surface of A431 cells was sensitiveto EndoH digestion (FIG. 48). This confirms that the form of EGFR boundby 806 on the cell surface of A431 cells is an EndoH sensitive form andthus is the high mannose type.

This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allaspects illustrated and not restrictive, the scope of the inventionbeing indicated by the appended Claims, and all changes which comewithin the meaning and range of equivalency are intended to be embracedtherein.

Various references are cited throughout this Specification, each ofwhich is incorporated herein by reference in its entirety.

TABLE 6  pREN ch806 LC Neo Vector Xho I 1CTCGAGAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTC 51ATTAGGCACCCCAGGCTTTACACTTTATGCTCCCGGCTCGTATGTTGTGT                                  EcoRI     EF1α promoter 101GGAGATTGTGAGCGGATAACAATTTCACACAGAATTCGTGAGGCTCCGGT 151GCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGG 201GGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAA 251ACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGG 301GGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAA 351CGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGC 401CTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACG 451CCCCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTG 501GGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCT 551TGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTG 601GCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAA 651ATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTA 701AATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGG 751CGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCC 801TGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGG 851CCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGC 901GGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGC 951TTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGA 1001GAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTC 1051AGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACC 1101TCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAG 1151GGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGT 1201TAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTG 1251AGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTT                    MluI       HindIII   PmeI 1301TTTCTTCCATTTCAGGTGTACGCGTCTCGGGAAGCTTTAGTTTAAACGCC 1351GCCACCATGGTGTCCACAGCTCAGTTCCTTGCATTCTTGTTGCTTTGGTTT        M  V  S  T  A  Q  F  L  A  F  L  L  L  W  F 1401CCAGGTGCAAGATGTGACATCCTGATGACCCAATCTCCATCCTCCATGTCTP  G  A  R  C  D  I  L  M  T  Q  S  P  S  S  M  S 1451GTATCTCTGGGAGACACAGTCAGCATCACTTGCCATTCAAGTCAGGACATT          V S L G D T V S I T C H S S Q D I 1501AACAGTAATATAGGGTGGTTGCAGCAGAGACCAGGGAAATCATTTAAGGGC N  S  N  I  G  W  L  Q  Q  R  P  G  K  S  F  K  G 1551CTGATCTATCATGGAACCAACTTGGACGATGAAGTTCCATCAAGGTTCAGT          L I Y H G T N L D D E V P S R F S 1601GGCAGTGGATCTGGAGCCGATTATTCTCTCACCATCAGCAGCCTGGAATCT          G S G S G A D Y S L T I S S L E S 1651GAAGATTTTGCAGACTATTACTGTGTACAGCATGCTCAGTTTCCGTGGACG E  D  F  A  D  Y  Y  C  V  Q  H  A  Q  F  P  W  T                                        BamHI 1701TTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGTGAGTGGATCCATCTGGG F  G  G  G  T  K  L  E  I  K  R 1751ATAAGCATGCTGTTTTCTGTCTGTCCCTAACATGCCCTGTGATTATGCGCAAA 1801CAACACACCCAAGGGCAGAACTTTGTTACTTAAACACCATCCTGTTTGCTTCTT 1851TCCTCAGGAACTGTGGCTGCACCA        T  V  A  A  P 1876TCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGC S  V  F  I  F  P  P  S  D  E  Q  L  K  S  G  T  A 1926CTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC  S  V  V  C  L  L  N  N  F  Y  P  R  E  A  K  V  Q 1976AGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC   W  K  V  D  N  A  L  Q  S  G  N  S  Q  E  S  V 2026ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC T  E  Q  D  S  K  S  T  Y  Y  S  L  S  S  T  L  T 2076GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCA  L  S  K  A  D  Y  E  K  H  K  V  Y  A  C  E  V  T 2126CCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG   H  Q  G  L  S  S  P  V  T  K  S  F  N  R  G  E        Nhe/Xba 2176TGTTGA GCTAGAACTAACTAACTAAGCTAGCAACGGTTTCCCTCTAGCGG  C  * 2226GATCAATTCCGCCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAA 2276TAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTC 2326TTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCA 2376TTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAAT 2426GTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTC 2476TGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCC 2526TCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAA 2576CCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCT 2626CTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCC 2676ATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACGTGTGTT 2751TAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTT 2801TTCCTTTGAAAAACACGATAATACCATGGTTGAACAAGATGGATTGCACG 2851CAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCA 2901CAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCA 2951GGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATG 3001AACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTT 3051CCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCT 3101GCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTC 3151CTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACG 3201CTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGA 3251GCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGG 3301ACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAG 3351GCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTG 3401CTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACT 3451GTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACC 3501CGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGT 3551GCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCC                    blunt end SalI/SalI 3601TTCTTGACGAGTTCTTCTGAGTCGATCGACCTGGCGTAATAGCGAAGAGG 3651CCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGG 3701GACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCG 3751CAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTT 3801TCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTA 3851AATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGA 3901CCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCT 3951GATAGACGGTTTTTCGCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTG 4001GACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTTA 4051TAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTA 4101ACAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGT 4151GGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATATTTGTTTATTTTTC 4201TAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAAT 4251GCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTG 4301TCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTACTGTTTTTGCTCAC 4351CCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACG 4401AGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTT 4451TTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTA 4501TGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCG 4551CCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAG 4601AAAAGCATATTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCC 4651ATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGG 4701AGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAA 4751CTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGAC 4801GAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACT 4851ATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACT 4901GGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCG 4951GCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCG 5001CGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAG 5051TTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAG 5101ATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA 5151AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTA 5201AAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCT 5251TAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAA 5301AGGATGTTCTTGAGATCCTTTTTTTCTGCACGTAATCTGCTGCTTGCAAA 5351CAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTAC 5401CAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAAT 5451ACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGT 5501AGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTG 5551CCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTA 5601CCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCC 5651CAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGC 5701TATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCG 5751GTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGG 5801AAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTG 5851AGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAAC 5901GCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGC 5951TCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA 6001CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGC 6051AGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCC 6101TCTCCCCGCGCGTTGGCCGATTCATTAATGCAGGTATCACGAGGCCCTTT 6151 CGTCTTCAC

The above nucleic acid sequence corresponds to SEQ ID NO:7. The aboveamino acid sequence, including any underlined amino acid sequencecorresponds to the sequence of SEQ ID NO:9.

TABLE 7  pREN 806 HC DHFR Vector Xho I 1CTCGAGAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTC 52ATTAGGCACCCCAGGCTTTACACTTTATGCTCCCGGCTCGTATGTTGTGT                                  EcoRI     EF1α promoter 102GGAGATTGTGAGCGGATAACAATTTCACACAGAATTCGTGAGGCTCCGGT 152GCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGG 202GGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAA 252ACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGG 302GGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAA 352CGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGC 402CTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACG 452CCCCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTG 502GGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCT 552TGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTG 602GCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAA 652ATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTA 702AATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGG 752CGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCC 802TGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGG 852CCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGC 902GGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGC 952TTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGA 1002GAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTC 1052AGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACC 1102TCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAG 1152GGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGT 1202TAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTG 1251AGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTT                   MluI        HindIII    PmeI 1302TTTCTTCCATTTCAGGTGTACGCGTCTCGGGAAGCTTTAGTTTAAACGCC 1352GCCACCATGAGAGTGCTGATTCTTTTGTGGCTGTTCACAGCCTTTCCTGGT      M  R  V  L  I  L  L  W  L  F  T  A  F  P  G 1401GTCCTGTCTGATGTGCAGCTTCAGGAGTCGGGACCTAGCCTGGTGAAACCT       V L S D V Q L Q E S G P S L V K P 1451TCTCAGACTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGT  S  Q  T  L  S  L  T  C  T  V  G  Y  I  T  S  T  S 1501GATTTTGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAAGCTGGAGTGG  D  F  A  W  N  W  I  R  Q  F  P  G  N  K  L  E  W 1551ATGGGCTACATAAGTTATAGTGGTAACACTAGGTACAACCCATCTCTCAAA  M  G  Y  I  S  Y  S  G  N  T  R  Y  N  P  S  L  K 1601AGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAATTCTTCCTGCAG  S  R  I  S  I  T  R  D  T  S  K  N  Q  F  F  L 1651TTGAATTCTGTGACTATTGAGGACACAGCCACATATTACTGTGTAACGGCG    

         

1701 GGACGCGGGTTTCCTTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA  G  R  G  F  P  Y  W  G  Q  G  T  L  V  T  V  S  A         BamHI 1751CAGTGAGTGGATCCTCTGCGCCTGGGCCCAGCTCTGTC 1801CCACACCGCGGTCACATGGCACCACCTCTCTTGCAGCCTCCACCAAGGGC                                      S  T  K  G 1851CCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACp  s  v  f  p  l  a  p  s  s  k  s  t  s  g  g  t 1901AGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG A  A  L  G  C  L  V  K  D  Y  F  P  E  P  V  T  V 1951TGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTs  w  n  s  g  a  l  t  s  g  v  h  t  f  p  a 2001GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCv  l  q  s  s  g  l  y  s  l  s  s  v  y  s  v  p 2051CTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGC S  S  S  L  G  T  Q  T  Y  I  C  N  V  N  H  K  P 2101CCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAA  S  N  T  K  V  D  K  K  V  E  P  K  S  C  D  K 2151ACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCT  H  T  C  P  P  C  P  A  P  E  L  L  G  G  P  S 2201AGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA V  F  L  F  P  P  K  P  K  D  T  L  M  I  S  R  T 2251CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG  P  E  V  T  C  V  V  V  D  V  S  H  E  D  P  E 2301GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAACGCCAAGACV  K  F  N  W  Y  V  D  G  V  E  V  H  N  A  K  T 2351AAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCC K  P  R  E  E  Q  Y  N  S  T  Y  R  V  V  S  V  L 2401TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAG  T  V  L  H  Q  D  W  L  N  G  K  E  Y  K  C  K 2451GTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCV  S  N  K  A  L  P  A  P  I  E  K  T  I  S  K  A 2501CAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGG K  G  Q  P  R  E  P  Q  V  Y  T  L  P  P  S  R  E 2551AGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC  E  M  T  K  N  Q  V  S  L  T  C  L  V  K  G  F 2601TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAY  P  S  D  I  A  V  E  W  E  S  N  G  Q  P  E  N 2651CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC N  Y  K  T  T  P  P  V  L  D  S  D  G  S  F  F  L 2701TCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTC  Y  S  K  L  T  V  D  K  S  R  W  Q  Q  G  N  V 2751TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAF  S  C  S  V  M  H  E  A  L  H  N  H  Y  T  Q  K                                        Nhe/Xba 2801GAGCCTCTCCCTGTCTCCGGGTAAATGAGCTAGAAACTAACTAAGCTAGC S  L  S  L  S  P  G  K  * 2851AACGGTTTCCCTCTAGCGGGATCAATTCCGCCCCCCCCCCCTAACGTTAC 2901TGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTAT 2951TTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGC 3001CCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGG 3051AATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTT 3101CTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCC 3151CCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATAC 3201ACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTG 3251TGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAG 3301GATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTG 3351CACATGCTTTACGTGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCC 3401GAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATAATACCATGGTT 3451CGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATTGGCAA 3501GAACGGAGACCTACCCTGGCCTCCGCTCAGGAACGAGTTCAAGTACTTCC 3551AAAGAATGACCACAACCTCTTCAGTGGAAGGTAAACAGAATCTGGTGATT 3601ATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAA 3651GGACAGAATTAATGGTTCGATATAGTTCTCAGTAGAGAACTCAAAGAACC 3701ACCACGAGGAGCTCATTTTCTTGCCAAAAGTTTGGATGATGCCTTAAGAC 3751TTATTGAACAACCGGAATTGGCAAGTAAAGTAGACATGGTTTGGATAGTC 3801GGAGGCAGTTCTGTTTACCAGGAAGCCATGAATCAACCAGGCCACCTCAG 3851ACTCTTTGTGACAAGGATCATGCAGGAATTTGAAAGTGACACGTTTTTCC 3901CAGAAATTGATTTGGGGAAATATAAACTTCTCCCAGAATACCCAGGCGTC 3951CTCTCTGAGGTCCAGGAGGAAAAAGGCATCAAGTATAAGTTTGAAGTCTA 4001CGAGAAGAAAGACTAACAGGAAGATGCTTTCAAGTTCTCTGCTCCCCTCC Blunt end SalI/SalI4051 TAAAGCTATGCATTTTTATAAGACCATGGGACTTTTGCTGGTCGATCGAC 4101CTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGC 4151GCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCG 4201GCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCT 4251AGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCG 4301GCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTT 4351AGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTC 4401ACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCTTTGACGTTGGA 4451GTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCA 4501ACCCTATCTCGGTCTATTTATAAGGGATTTTGCCGATTTCGGCCTATTGG 4551TTAAAAAATGAGCTGATTTAACAAAATTTAACGCGAATTTTAACAAAATA 4601TTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACC 4651CCTATATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATG 4701AGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTAT 4751GAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTT 4801GCCTTACTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCT 4851GAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAG 4901CGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGA 4951GCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCC 5001GGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGT 5051TGAGTACTCACCAGTCACAGAAAAGCATATTACGGATGGCATGACAGTAA 5101GAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAAC 5151TTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCA 5201CAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGA 5251ATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATG 5301GCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTC 5351CCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCAC 5401TTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGA 5451GCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGG 5501TAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTA 5551TGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAG 5601CATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTT 5651AAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATA 5701ATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCA 5751GACCCCGTAGAAAAGATCAAAGGATGTTCTTGAGATCCTTTTTTTCTGCA 5801CGTAATCTGCTGCTTGCAAACAAAAAACCACCGCTACCAGCGGTGGTTTG 5851TTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCA 5901GCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGC 5951CACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAAT 6001CCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGT 6051TGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG 6101GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACT 6151GAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGA 6201GAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGC 6251ACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGG 6301GTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGG 6351GGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTG 6401GCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGA 6451TTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCC 6501GCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAG 6551CGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG 6601CAGGTATCACGAGGCCCTTTCGTCTTCAC

The above nucleic acid sequence corresponds to SEQ ID NO:8. The aboveamino acid sequence, including any underlined amino acid sequencecorresponds to the sequence of SEQ ID NO:10.

What is claimed is:
 1. An isolated antibody produced by hybridoma cellline 806 (ATCC Accession No. PTA-3858).
 2. The hybridoma cell line 806deposited under ATCC Accession No. PTA-3858.